Method for incubation or processing of samples, biocompatible device for vibroacoustic control and use of same

Abstract

The invention relates to a method for incubation or processing of samples, in one or more containers inside an incubator, comprising a step of inserting the container with the samples into a biocompatible device for vibroacoustic control. The invention also relates to a biocompatible device for vibroacoustic control, formed by a receptacle with an internal base for the samples, with means for accessing the interior of the receptacle, supporting means on the internal surface of the incubator and means for controlling the environment, including perforations in the walls of the receptacle, and the incubation of the samples inside the biocompatible device for vibroacoustic control.

Description

[0001] DESCRIPTION

[0002] Sample incubation or processing procedure, biocompatible vibroacoustic control device and use

[0003] FIELD OF INVENTION

[0004] The present invention belongs to the biological sector and more specifically to the field of cell culture and the manipulation or processing of cultures in general.

[0005] The main object of the present invention is a sample incubation and processing procedure that incorporates a biocompatible vibroacoustic device. This device provides a convenient, fast, and safe way to reduce mechanical stress on biological samples, improving the reliability of cell dynamics assays. It is applicable in various contexts, such as incubators, biological safety cabinets (especially laminar flow hoods), or any environment where the sample is subject to noise or vibration.

[0006] STATE OF THE ART

[0007] Noise is a constant presence in all aspects of modern life. Currently, there is a general consensus in the scientific community regarding the negative impact that continuous noise exposure has on people's physical and mental health (Bistrup, 2003 [2]; Bradley & Lang, 2000 [3]; Münzel et al., 2018 [6]). However, noise, due to its mechanical wave nature, also exerts a direct influence on fundamental processes at the cellular level (Kumeta et al., 2018 [4]; Nikukar et al., 2013 [8]).

[0008] Mechanobiology is an emerging discipline dedicated to the study of cellular dynamics in response to various mechanical stimuli, including audible acoustic waves (Ambattu & Yeo, 2023 [1]; Kumeta et al., 2018 [4]; Nikukar et al., 2013 [8]). The mechanotransduction processes promoted by these stimuli play a crucial role in regulating cellular homeostasis, being determinants in aspects as relevant to medicine as wound healing, neurodegenerative diseases, and cancer (Leí et al., 2023 [5]; Tassinah et al. [9], 2023; Tortorella et al., 2022

[0010] ). Several studies in the field of mechanobiology demonstrate the influence of noise on cellular dynamics (Kumeta et al., 2018 [4]; Navarro-Fernández, 2010 [7]).

[0009] At the same time, cell culture incubators are essential equipment in biological laboratories, maintaining constant, optimal conditions for cell growth and dynamics through controlled systems (Triaud et al., 2003

[0011] ). These controlled systems employ electromechanical actuators, ventilation systems, valves, and compressors that generate vibrations and audible noise, which can interfere with the reliability of cell dynamics studies in conventional cell culture incubators.

[0010] Therefore, regulatory systems affect the samples, but they depend on the specific equipment used. This reduces the reproducibility of the studies, since each laboratory has different equipment or even defines different ways of maintaining assay conditions. It is necessary to reduce this variability, but the applicant is unaware of any device that protects cell samples from the noise generated by incubators and other equipment, leaving the cultures exposed to their effects.

[0011] [1] Ambattu, LA, & Yeo, LY (2023). Sonomechanobiology: Vibrational stimulation of cells and its therapeutic implications. Biophysics Reviews, 4(2), 21301. https: / / doi.org / 10.1063 / 5.0127122 / 2884904

[0012] [2] Bistrup, M. L. (2003). Noise and Health. Noise and Health, 5(19), 59. https: / / www.noiseandhealth.org / article.asp?issn=1463- 1741 ;year=2003;volume=5;issue=19;spage=59;epage=64;aulast=Bistrup

[0013] [3] Bradley, M. M., & Lang, P. J. (2000). Affective reactions to acoustic stimuli. Psychophysiology, 37(2), 204-215. https: / / doi.org / 10.1111 / 1469-8986.3720204

[0014] [4] Kumeta, M., Takahashi, D., Takeyasu, K., & Yoshimura, S. H. (2018). Cell type-specific suppression of mechanosensitive genes by audible sound stimulation. PLOS ONE, 13(1), e0188764. https: / / doi.org / 10.1371 / JOURNAL.PONE.0188764

[0015] [5] Lei, Z., Jiang, H., Liu, J., Liu, Y., Wu, D., Sun, C., Du, Q., Wang, L, Wu, G., Wang, S.,

[0016] & Zhang, X. (2023). Audible Acoustic Wave Promotes EV Formation and Secretion from Adherent Cancer Cells via Mechanical Stimulation. ACS Applied Materials and Interfaces, 15(46), 53859-53870. https: / / doi.org / 10.1021 / ACSAMI.3C13845 / SUPPL_FILE / AM3C13845_SI_001.PDF

[0017] [6] Münzel, T., Schmidt, F.P., Steven, S., Herzog, J., Daiber, A., & Sorensen, M. (2018). Environmental Noise and the Cardiovascular System. Journal of the American College of Cardiology, 71(6), 688-697. https: / / doi.Org / 10.1016 / J.JACC.2017.12.015

[0018] [7] Navarro-Fernández, N. (2010). Characterization and quantification of the influence of music as a physical agent on the behavior of embryonic neural stem cells in culture [University of Valladolid], https: / / dialnet.unirioja.es / servlet / tesis?codigo=295435&info=resumen&idioma=SPA

[0019] [8] Nikukar, H., Reid, S., Tsimbouri, PM, Riehle, MO, Curtis, ASG, & Dalby, MJ (2013). Osteogenesis of mesenchymal stem cells by nanoscale mechanotransduction. ACS Nano, 7(3), 2758-2767. https: / / doi.org / 10.1021 / NN400202J

[0020] [9] Tassinari , R. , Olive , E. , Cavallini , C. , Taglioli , V. , Zannini , C. , Marcuzzi , M. , Fedchenko , O. , & Ventura , C. (2023). Mechanobiology: A landscape for reinterpreting stem cell heterogeneity and regenerative potential in diseased tissues. / Science, 26(1), 105875. https: / / doi.org / 10.1016 / J. ISCI .2022.105875

[0021]

[0010] Tortorella , I. , Argentati , C. , Emiliani , C. , Morena , F. , & Martino , S. (2022). Biochemical Pathways of Cellular Mechanosensing / Mechanotransduction and Their Role in Neurodegenerative Disease Pathogenesis. Cells, 77(19). https: / / doi.org / 10.3390 / cells11193093

[0022]

[0011] Triaud , F. , Clenet , DH , Cariou , Y. , Neel , T. Le , Morin , D. , & Truchaud , A. (2003). Evaluation of Automated Cell Culture Incubators. SLAS Technology, 8(6), 82-86. https: / / doi.org / 10.1016 / S1535-5535(03)00018-2

[0023] EXPLANATION OF THE INVENTION

[0024] The invention's procedure improves reproducibility and reliability in assays by incorporating a biocompatible noise and vibration control device. This device comprises a container capable of reducing noise levels generated by traditional incubators, thereby increasing reliability in cell dynamics studies in a convenient, fast, and safe manner.

[0025] The main advantages of the invention are summarized as follows:

[0026] • Reduces mechanical stress at the cellular level by attenuating airborne and impact noise on the culture plate.

[0027] • Reduces vibration transmissions by decoupling the culture plate from the incubator structure.

[0028] • It improves the homogeneity of environmental conditions by reducing vibroacoustic fluctuations and variations in the cellular environment. Therefore, the test results become independent of the equipment used and its configuration. • It increases the consistency and reproducibility of cell dynamics experiments by minimizing masking phenomena caused by ambient vibroacoustic energy.

[0029] • Improves sensitivity in cell and molecular dynamics assays by reducing the mechanical stress that background noise from the incubator exerts on the biological sample.

[0030] • It optimizes available space by allowing stacking. This stacking of different devices, in turn, increases the equivalent sound absorption area and results in a greater reduction of overall cellular stress.

[0031] The procedure for incubating and processing samples in one or more containers within an incubator follows the standard steps for this type of procedure, depending on the research objective. Thus, in an incubator, the typical procedure includes the phases of seeding, incubation, experimentation, and sample processing. It also includes a step of inserting the container with the samples into a biocompatible vibroacoustic control device. This device consists of a container with an internal base for the samples, access points, support structures on the incubator's inner surface, and environmental control mechanisms that include perforations in the container walls. Sample incubation is then carried out within the biocompatible vibroacoustic control device.

[0032] The invention also relates to the biocompatible vibroacoustic control device usable in the procedure and to the use thereof.

[0033] The biocompatible vibroacoustic control device used in the invention comprises a sample container, access means, support means, and environmental control means, consisting primarily of perforations in the main body and access means, as well as the material from which they are made. Environmental control means may also be located on the side walls.

[0034] The main body is made of a material with optimal acoustic performance, especially in the audible spectrum, although other frequency ranges may also be advantageous. Specifically, it has a sound absorption coefficient between 0.5 and 0.99. An example is the panel marketed under the brand name ECOcero panel. For example, absorption coefficients are 0.52 at 500 Hz, 0.72 at 1 kHz, and 0.99 at 4 kHz. Depending on the application, the material may need to be biocompatible to ensure that sterile conditions are maintained at all times, even with accidental contact. This means a material with low release of volatile organic compounds and antifungal properties. An example is the "ECOcero panel" material from the company ECOcero (Spain). It is a hollow structure or container with a flat base on which the growing plate is placed. It can have different sections or accommodate different plants, but a rectangular or square shape is preferred.

[0035] The support means are integrated with the main body and are intended to decouple the vibroacoustically device from the structure of the incubator or other equipment that contains it.

[0036] Environmental regulation means comprise perforations or through-holes. Their main function is to promote thermodynamic equilibrium and gas exchange between the device of the invention and the incubator.

[0037] In a preferred embodiment, the thickness of the walls, access means, and container walls can vary between 5 mm and 20 cm. Greater thickness of the main body results in a higher absorption coefficient and lower biological stress inside.

[0038] In another preferred embodiment, the main body can take the form of a cube, rectangular prism, square or triangular-based pyramid, truncated or not, cone, cylinder, triangular prism, tetrahedron, dodecahedron, octahedron or any other configuration that can be stably supported on the inner surface of the equipment.

[0039] In another preferred embodiment, the access means can have an area of ​​between 5% and 100% of the base surface.

[0040] In another preferred embodiment, the means of access to the container can be of one piece or of several pieces that allow access to different parts of the container, their independence from the main body can be partial (hinged) or total (detachable) and the closure of the access means can be carried out by tongue and groove, adhesive, interlocking assembly, screws, nuts, welding, groove, tabs, Velcro, clips or other types of fastenings.

[0041] In another preferred embodiment, the surface area of ​​the support media in contact with the incubator can range from 1% to 100% of the device's footprint. The smaller this surface area, the less vibration is transmitted between the device of the invention and the incubator structure. Consequently, there is less mechanical stress on the biological sample.

[0042] In another preferred embodiment, the perforations cover between 1% and 40% of the surface area of ​​the base and access means, and can adopt different configurations in terms of number, location, and shape. The number of perforations ranges from one perforation representing 1% of the total area to an indeterminate number such that the sum of the perforation surfaces is less than 40% of the total area of ​​the device of the invention. The location of the environmental control means can be distributed on any surface of the biocompatible noise control device of the invention. The shape of the environmental control means can employ any known geometry, provided it meets the surface conditions described.The relationship between the environmental control measures and the total surface area of ​​the device of the invention will determine the total absorption area, influencing the mechanical stress on the biological sample. Ideally, the perforations are labyrinthine, with curves and / or bends, to limit sound entry.

[0043] In another preferred embodiment, the main body, access means, support means, and side walls may be separate elements and assembled by tongue and groove, adhesive, interlocking assembly, screws, nuts, welding, grooves, tabs, Velcro, clips, or other fasteners. They may also be manufactured as a single, indivisible piece.

[0044] Ultimately, another preferred embodiment can also be considered, incorporating an active control system to selectively attenuate a specific band of the spectrum within the device of the invention. This active control would utilize electroacoustic transducers, allowing the band to be selected by configuring or adjusting them.

[0045] The device allows biological samples to be preserved in suitable plates or containers, isolated from noise and vibrations, for the study of cell dynamics.

[0046] BRIEF DESCRIPTION OF THE FIGURES

[0047] To complement the description made and in order to facilitate the understanding of the characteristics of the invention, according to a preferred example of its practical embodiment, a set of figures is included as an integral part of said description in which, for illustrative and non-limiting purposes, the following has been represented: Figure 1 shows a perspective view of an example of the device that is the subject of the invention.

[0048] Figure 2 shows an exploded view of the embodiment of Figure 1.

[0049] Figure 3 shows a perspective view of the device in Figure 1 with the access medium open.

[0050] Figure 4 shows a side view of the device in Figure 1 with the access means open.

[0051] Figure 5 shows a view of the underside of the device in Figure 1.

[0052] DETAILED EXPOSURE OF MODES OF REALIZATION

[0053] In view of the figures cited, and in accordance with the numbering adopted, an example of a preferred embodiment of the invention can be observed, which comprises the parts and elements that are indicated and described in detail below.

[0054] The incubation and handling or processing procedure of the invention begins by placing the cell samples in a container, such as a Petri dish, and this container in a biocompatible vibroacoustic control device. This device is then placed in the corresponding incubator or handling or processing equipment.

[0055] Figures 1 to 5 show a possible preferred embodiment of the device of the invention. It comprises a container (1), preferably with a rectangular base to optimize space in equipment (incubators, etc.). In this embodiment, the container (1) consists of a main body (2) with a general U-shape and two side plates (3), leaving an upper opening where a lid or access means (4) is placed. The main body (2) and the side plates (3) can be assembled by means of tabs (10) that fit into grooves (11).

[0056] The upper access means (4) has a handle (7) that fits into a locking groove (8) and is retained by friction. The interior of the container (1) comprises a base (9) for placing the samples. The lid (4) and the base (9) have perforations (6) to allow communication between the contents of the device and the external environment, thus promoting thermodynamic equilibrium and gas exchange between the device of the invention and the incubator. The base (9) is separated from the support surface by legs or support means (5) that decouple the base (9) from the vibration of the support surface. This decoupling can be achieved by reducing the contact area with the incubator or the floor of the bell jar, or elastic layers can be arranged in contact with the support surface to improve isolation.

[0057] With this preferred embodiment, the reduction of incident acoustic energy on the culture plate inside the device of the invention is 20 dB at 500 Hz, 31 dB at 1 kHz, and 48 dB at 5 kHz for a standard airborne signal-to-noise ratio (MLS) measurement. Simultaneously, in a cell dynamics assay, significant differences were obtained for the same cell line and seeding / incubation conditions (p < 0.001), with averages of 0.735 ± 0.100 and 0.644 ± 0.183 for a culture plate located inside the device of the invention and the incubator tray, respectively.

[0058] The preferred embodiment achieving the stated results is a device made of ECOcero material with a total width of 148.00 mm, a main body (2) 124.00 mm wide and 12.00 mm thick. Its tabs (10) and slots (11) are 20.00 mm wide. The total height of the side plate (3) is 85.50 mm. This plate comprises support means (5) 12.50 mm wide with a 5.00 mm radius cut and a main body (2) 63.00 mm high. The lid (4) spans 170.00 mm, and the overall length of the preferred embodiment is 194.00 mm. The perforations in the lid (6) and base (9) are 6.89 mm in diameter, 12.00 mm thick, and evenly spaced.

[0059] A cell proliferation assay is described below:

[0060] Cells from the 661W cell line were seeded in the culture plate, which was subsequently introduced into the device of the invention. This cell line, kindly provided by Dr. Muayyad Al-Ubaidi of the University of Oklahoma (USA), is derived from a transgenic muhna retina and shows the expression of photoreceptor markers and retinal ganglion cell markers (Sayyad et al., 2017; Tan et al., 2004).

[0061] Before each assay, cells were seeded at a density of 100,000 cells per flask and incubated at 37 °C with 5% CO2. The medium used was Dulbecco's Modified Eagle Medium (DMEM) composed of 4.5 g / L glucose, supplemented with 2 mM L-glutamine, penicillin / streptomycin, and 10% (v / v) heat-inactivated fetal bovine serum.

[0062] Cell proliferation was assessed using the crystal violet assay (Mickuviene et al., 2004). Cells were seeded in sterile 96-well polystyrene plates (SPL Life Sciences, Pocheon-si, South Korea) at a density of 3,000 cells per well (40 replicates) and incubated for 24 hours (Tuohy et al., 2002). Subsequently, the plates were placed in the incubator (Forma™ Steh-Cycle™ CO2 incubator, model 371, Thermo Electron Corporation, Waltham, MA, USA) where they were maintained for 72 hours. The study was conducted under two experimental conditions: (i) the "control" condition, where the biological samples were exposed to the noise generated by the electromechanical systems and actuators within the incubator (Triaud et al., 2003), and (ii) the "box" condition, in which the biological samples were placed inside the device of this invention. Three independent replicates were performed.

[0063] The cells were fixed using 96% (v / v) ethanol for 10 minutes and then stained with a 0.05% (w / v) crystal violet solution in 20% (v / v) ethanol for 30 minutes. The plates were then washed three times with distilled water and air-dried at room temperature in a light-protected environment. To dissolve the stain, 100 µL of 50% (v / v) ethanol and a 0.1% (v / v) acetic acid solution were introduced into each well of a plate and incubated at room temperature for 5 minutes. Finally, the supernatant was transferred to a sterile 96-well plate and the absorbance was quantified using a spectrophotometer (AD 340 Microplate Reader, Beckman Coulter, Brea, CA, USA) at a wavelength of 620 nm.

[0064] Regarding the results, the absorbance values ​​under the "control" and "box" conditions were 0.571 ± 0.174 and 0.690 ± 0.153, respectively. Sample normality was verified using the Kolmogorov-Smirnov test, and the values ​​were compared using a two-sample t-test, yielding statistically significant results (p < 0.0001). The results obtained demonstrate a statistically significant increase in cell proliferation in the "box" condition compared to the "silence" condition.

[0065] Acoustic measurements were performed using a laptop computer with ARTA software (version 1.9.1) connected to a TASCAM 144 mk2 acquisition card and a Brüel&Kjaer Type 1704-A-002 2-channel CCLD signal conditioner feeding a Brüel&Kjaer Type 4188-A-21 microphone. A Bose SoundLink Color II loudspeaker suspended on a perch inside the incubator served as the acoustic source. Ultimately, the stimulus employed was a Maximum Length Sequence (MLS) signal, and the Sound Pressure Level was recorded.

Claims

CLAIMS 1. A method for incubating or processing samples in one or more containers, characterized in that it comprises a step of inserting the container with the samples into a biocompatible vibroacoustic control device formed by a container (1) with an inner base (9) for the samples, with access means (4) to the interior of the container, support means (5) on the inner surface of the incubator and environmental regulation means including perforations (6) in the walls of the container, and the incubation or processing of the samples within the biocompatible vibroacoustic control device.

2. Biocompatible vibroacoustic control device, usable in the procedure of claim 1, characterized in that it is formed by a container (1) with an inner base (9) for the samples, with access means (4) to the interior of the container (1), support means (5) and environmental regulation means including perforations (6) in the walls of the container.

3. Biocompatible vibroacoustic control device, according to claim 2, characterized in that the environmental regulation means are arranged in the base (9) and the access means (4).

4. Biocompatible vibroacoustic control device, according to claim 2, characterized in that it incorporates an active control system to selectively attenuate a specific band of the spectrum inside the container (1).

5. Biocompatible vibroacoustic control device, according to claim 2, characterized in that the perforations (6) are labyrinthine.

6. Use of the biocompatible vibroacoustic control device of claim 2 for insertion of biological sample containers during their incubation or processing

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