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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/22—Transparent or translucent parts
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/20—Material Coatings
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/02—Form or structure of the vessel
  • C12M23/10—Petri dish
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/02—Form or structure of the vessel
  • C12M23/16—Microfluidic devices; Capillary tubes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/06—Plates; Walls; Drawers; Multilayer plates

Definitions

• the present invention relates to materials and methods for culturing biological samples.
• it relates to supports for cell cultures which are adapted to deliver heat for the said samples in a highly precise and controllable way so to induce differentiation of cells.
• Culturing of biological samples is generally performed to develop them from a precursor to mature stage and / or to propagate and grow them in mass, dimension, surface, etc.
• Typical cultured biological samples are cells, cell aggregates, unicellular or multicellular organisms, tissues, parts of organs (biopsies), etc.
• One important issue in culturing methods is the rate of growth and differentiation: often this is achieved by the addition of proper culture media with growth or differentiating factors and by keeping the temperature of the biological sample close to 37 ° C. Other physical stimuli than temperature have been rarely applied to stimulate cell differentiation.
• the temperature control is an important issue in culturing methods: in fact, biological samples are temperature-sensitive and their viability and functionality are safeguarded within limited temperature ranges.
• very few studies (Shui et al. 2001 1 ; Hossain 2017 2 ; Wang 2016 3 ; Oyama 2015 4 ; Kudo 2015 5 , to name a few) show that the application of specific thermal stimuli, possibly periodically repeated, to different cell lines in culture can improve the culturing yield, in terms of differentiation and/or growth.
• Two types of thermal applications can be distinguished: underlying ones and stimulating ones.
• the standard underlying thermal application is essentially aimed at safeguarding the viability/ functionality of the biological sample; it involves setting a stable incubation temperature throughout the culturing period, which is normally applied by the whole reactor in which the incubation takes place.
• stimulating thermal applications are typically episodic, intermittent, or cyclic treatments performed during and in addition to the normal temperature incubation: they are characterized by a transient/ sudden temperature change, typically an increase, with respect to the incubation temperature; these thermal events should be much shorter in duration compared to the overall incubation time and are possibly sharp in their rate of establishment and disappearance.
• a basic way to perform stimulating thermal applications consists in modifying the general temperature of the incubator. This method is however not practical because of the inertia of the apparatus, particularly in case of large-sized industrial incubators. These apparatuses do not allow a sharp and controlled temperature increase to be applied to the biological sample, such that the target temperature is reached too slowly; trying to increase the heating rate by applying a higher temperature to the incubator is not a valid solution, as it may cause overheating of the biological sample, with consequent loss of viability and/or functionality of the cells in the sample (thermal inertia too large) . Stimulating thermal treatments are more conveniently applied separately/ independently from the underlying incubation temperature, via additional heating means, for example heating lamps, i.e.
• infrared lamps located inside the incubator at a close distance from the biological sample.
• Such additional means can apply more rapid and controllable thermal events to the cultured sample.
• the corresponding processes result in an inconsistent or insufficient stimulation for the specific portion of the sample to be treated and possibly even in a detrimental effect on the portion of the sample that one does not wish to treat, which translates into less effective results on the culture.
• US 2020/0318053 describes a cells sorter constituted by a microfluidic channel suitable to recover free cells from the cell culture medium; the container includes a gel layer which contains gold nanoparticles dispersed in bulk; the cell culture comes in direct contact with the gel; on operation, the nanoparticle gel layer is subjected to irradiation, it releases heat via photothermic effect and is denaturated, weakening adhesion of the selected cell, whereby the said cell is released in the free state and is removed from the container by fluxing a suitable liquid.
• US 2020/0017815 describes a cell sorter constituted by a cell culture substrate comprising two fluid gel layers, wherein gold nanoparticles are present in the first layer and, optionally also in the second layer at a concentration lower than in the first layer; cells are seeded on an area of the first layer not overlapping with the second layer; light is transmitted to this area, which causes the gel to heat, denaturate and release the selected seeded cell in the free state.
• US202 1/032584 describes a blood vessel-mimicking microfluidic chip in which cells to be co-cultured are vascular endothelial and cancer cells to mimic a vascularized tissue, eventually cancer-metatastic. Nanoparticles, e.g. gold nanoparticles, can be incorporated in the cell culture in order to study photothermal therapy on cancer cells.
• WO2019/ 185731 describes a flexible film with thermal effect upon light irradiation, containing Gold nanostars or Prussian blue nanoparticles; the film is used for manufacturing a fast-responding thermal patch for application to the skin resulting in therapeutic heat with pain relief properties.
• organoid is a 3D multicellular in vitro tissue that mimics its corresponding in vivo organ, such that it can be used to study aspects of that organ in a culture dish.
• 3D tissue culture is decades old, today the word organoid is commonly used to describe such constructs derived from stem cells; these could be either pluripotent (embryonic or induced) or adult stem cells from various organs. It is thought that the processes that form these tissues in vitro approximate natural development and/or tissue maintenance.
• one approach to generate brain organoids embeds neuroectodermal embryoid bodies from human pluripotent stem cells (hPSCs) in Matrigel, but gives no further external cues to the cells.
• hPSCs human pluripotent stem cells
• This approach reproduces many brain regions, including cortex, but it can also generate some mesodermal and endodermal lineages.
• cells are patterned toward more specific regions of the CNS, typically based on prior knowledge of the signals controlling development, with further contribution from self-organizing processes.
• region-specific brain organoids are generated by patterning hPSCs towards structures that consist principally of either dorsal or ventral forebrain.
• growth conditions typically mimic signals that control tissue repair after damage or steady state tissue maintenance.
• epithelial organoids derived from adult stem cells of the gastrointestinal tract need agonists of Wnt signaling and other signaling factors embedded in the cell culture to both maintain the cells and to generate an in vivo-like complement of cell types.
• the present invention aims at efficient means and methods to apply stimulating thermal treatments to cell cultures, including complex ones, i.e. treatments which require a diversified over time, and/or sharply defined, and/or localized applications of heat, also with a view to the production of non-homogeneously grown cell cultures and organoids derivable therefrom. It also aims at improving the efficiency and reproducibility of heating stimulation applied to the cell culture; it also aims at providing methods for differentiating with high efficiency specific cell types, such as for example neurons.
• cell cultures in particular neuronal cell cultures, can be selectively induced in their differentiation by a localized heat induction.
• the present inventors have now found that an efficient thermal stimulation of a cell culture can be reached at high spatial resolution, being able to deliver the temperature in a more focused pattern, even at the level of single cells.
• Such patterned heating was obtained by the present inventors for the first time by incorporating nanoparticles provided with photo-thermal effect which can be induced by irradiation with wavelength between 0.4 pm and 1.2 pm in those specific areas of the support where the thermal treatment is required. As shown by the experiments reported in this description, such patterned heating was found unexpectedly efficient in stimulating neuronal differentiation.
• Object of the invention is therefore a support for cell culture comprising, incorporated therein or layered beneath, nanoparticles provided with photo-thermal effect.
• the support is optically transparent and, together with said layered or incorporated nanoparticles, forms a rigid structure which can be shaped in the form of standard containers for the incubation of biological samples such as a Petri dish, etc., and is made of materials compatible therewith, typically glass, plastics.
• the nanoparticles are disposed in selected portions of the support in correspondence of the support surface aimed at holding the cell culture.
• the nanoparticles are layered on the support: the layering is applied on the support surface being opposite to the one coming into contact with the cell culture, i.e. beneath the support, thus avoiding a direct physical contact between nanoparticles and cell culture: for example, if the support is a Petri dish, the nanoparticle layer will be applied only to the external lower surface of the dish, excluding layering on the upper dish surface which comes in direct contact with the cell culture.
• the layering can be obtained by coating the support with a nanoparticle-containing polymeric material, wherein the polymer can be chosen for example among polyvinyl alcohol, polyvinyl pyrrolidone and/or chitosan.
• a further object of the invention is a method of manufacturing the above- mentioned support, comprising layering beneath /incorporating in a suitable support precursor, the above-mentioned nanoparticles.
• a further object of the invention is a method of using the support, i.e. a method of culturing cells and obtaining cell differentiation, comprising: (i) placing a cell culture in contact with the support and (ii) irradiating the support with wavelength between 0.4 pm and 1.2 pm. The method can be used e.g. to induce a highly efficient differentiation of a neuronal cell culture
• Figure 1 Representative images of F-l 1 cells maintained at 37°C (A) and thermally stimulated at 41.5°C (B). Cells at 41.5°C showed significantly longer neurites (black arrows) compared to the control, suggesting that the remote thermal stimulation was able to modify cell morphology. Images were taken four days after completing the heating protocol (30 minutes at 41.5°C on days 1 and 2).
• FIG. 3 Functional analysis of F-l 1 cell electrophysiological properties by the Patch-Clamp technique on days 7 and 8 confirmed a more differentiated profile for stimulated cells (IR) versus 37°C (CTRL).
• cell culture used herein is not limited to specific cell types, and generally comprises any of them, also including stem cells.
• the cell culture is a neuronal cell culture or a neural stem cell culture; the culture medium will be conformed to the used cells.
• differentiation is used herein to indicate the evolution of the cell population of the cell culture to a fully mature status, from the morphological and / or functional point of view, wherein such evolution is reflected by a modified measurable property of the cell, e.g. neurite elongation and/or electrical activity, in the case of a culture of neuronal cells.
• highly efficient differentiation or “differentiation with high efficiency” means the capacity to obtain a high level of differentiation, as above defined, in a limited incubation time and without the need of adding differentiation agents; in particular, when applied to neuronal cell cultures, a highly efficient differentiation can be measured as obtaining a high values of neurite elongation and/or electrical activity after a limited incubation time and without the need of adding differentiation agents.
• support refers to any solid material which is suitable to maintain in a stable position a cell culture placed thereon.
• the support inclusive of the layered or incorporated nanoparticles, is rigidly structured, i.e. has a Young’s modulus of 0.4 MPa or higher, for example between 0.4 and 1.2 MPa.
• the support is thus structurally different from e.g. flexible patches, films and the like.
• the structural rigidity of the support is obtained by using rigid materials such as glass, plastics and the like as composite materials of the support.
• the support is also optically transparent. It is mainly container-shaped or plate-shaped, depending on the characteristics of the cell culture to be supported, e.g. samples in suspension, or adherent samples.
• Typical, non-limitative forms of the support used in the invention are a Petri dish, a tube, a slide (e.g. a microscope slide), a tray, a flask, a reaction vessel also in the form of a micro-capillary, and microfluidic chambers etc.
• the support surface comprises areas aimed (e.g. shaped and/or marked for this function) at coming into contact with the cell culture: these areas can be e.g. the bottom part of a container, the central part of a plate, etc.; the nanoparticles can be present in correspondence of selected portions of said areas, where a localized thermal treatment is to be applied.
• said selected portions taken as a whole, cover less than 80%, or less than 50%, or less than 40%, or less than 30%, or less than 20%, or less than 10% of the area of the support which is aimed at coming into contact with the cell culture.
• the selected portions containing the nanoparticles can be shaped and positioned as desired within the area of the support which is aimed at coming into contact with the cell culture, to obtain a localized heating and a differential stimulation of the cell growth and / or differentiation in the corresponding areas of the cell culture, compared to the remaining areas of the cell culture.
• the selected portions can be, for example, stripe-shaped, dot-shaped, triangle-shaped, square-shaped, rectangle-shaped and/or ellipse shaped, combinations of the same, etc.
• the selected portions can form, as a whole, a pattern forming the scaffold of a biological structure or sub structure, for example of one or more neurites or ganglia, as possible elements of a neural organoid.
• correspondence of used herein to define the positioning of the nanoparticles excludes a direct, i.e. physical contact between the nanoparticles and the cell culture itself; in particular, the term “in correspondence” defines a localization of the nanoparticles at a close vicinity to the cell culture, typically at a 140-650 or more micrometers distance from the cell culture, for example 140-300 micrometers or 140-170 micrometers; therefore, a nanoparticle-free section of the support always remains, separating the nanoparticles from a direct physical contact with the cell culture.
• the nanoparticles are present at concentrations generally comprised between 0.1 and 20 nanoparticles/ pm 3 ; they can be further varied and/or optimized according to the specific materials in use.
• photo-thermal effect relates to the capacity of the nanoparticles to release heat when irradiated at the selected wavelength, typically within the visible or near infrared spectrum. Suitable wavelengths range from 0.4 pm to 1.2 pm, preferably from 0.5 pm to 1.0 pm, more preferably from 0.6 pm to 0.9 pm. Irradiation can be supplied by any suitable device, e.g. a lamp, emitting visible and/or NIR light in the above stated wavelength ranges. Special irradiating devices, also usable in the invention are LED-based ones; among them, particularly interesting are those equipped with optical systems enabling to direct and change the shape of the irradiation area. Examples of these devices are described in the patent application WO2019185731, herein incorporated by reference.
• Nanoparticles with photo-thermal effect usable in the present invention are widely known per se and are commercially available; they can be used singly or as mixtures of different nanoparticles.
• nanoparticles are used which have an efficiency of conversion between absorbed radiation and emitted heat (herein also measured as Specific Adsorption Rate) higher than 50 kW/g, particularly between 50 and 300 kW/g, preferably between 150 and 300 kW/g.
• Specific Adsorption Rate (conventionally referred to as SAR) is defined as: wherein C is the thermal capacity of the suspension and MNP is the total mass of the nanoparticles.
• suitable nanoparticles are Gold Nanostars (GNSs), pegylated Gold Nanostars and/or Prussian Blue nanoparticles (PBNPs) . Due to their high SAR values, these nanoparticles are characterized by a very short induction time (onset of the photo- thermal response), i.e. reaching the desired temperature typically within 5 s from the beginning of irradiation. Said GNSs and PBNPs are also capable to reach a stable plateau of temperature, which lasts during the whole irradiation time. GNSs are commercially available e.g. from NanoSeedz and NanoimmunoTech
• GNSs and PBNPs are biocompatible and nontoxic; PBNPs are also approved by the U.S. Food and Drug Administration (FDA). If desired, GNSs can be produced by known synthetic methods, which include using the surfactant Triton X-100 (see e.g. Pallavicini et al., Chem.Commun., 2013, 49, 6265-6276, herein incorporated by reference).
• LSPR localized surface plasmon resonances
• the PBNPs can be obtained by means of known procedures (see, e.g., e.g.
• nanoparticle size can be widely varied within the nano range: non-limitative values may range from 5 to 100 nm.
• the present invention extends to a method of manufacturing a support for cell culture as above described, comprising layering on or incorporating in a suitable support precursor, nanoparticles provided with a photo-thermal effect which can be induced by irradiation with wavelength ranging from 0.4 mih to 1.2 mih.
• support precursor means herein a support not containing the nanoparticles.
• the support precursor is typically used at the fluid or gel-like state; the fluidity of the support material is typically obtained via heating.
• the viscosity and the surface tension of the support material can be varied by addition of low molecular weight alcohols or polymers, like PEG or PVP.
• the nanoparticles are introduced into selected internal portions of the previously formed support.
• the incorporation can be obtained, for example, by casting a molten, nanoparticle-free mass of the support material into its final shape and then, prior to final solidification, injecting the nanoparticles into selected internal portions of the support mass.
• the nanoparticles can be introduced into the finally solidified support: in this case, suitable spaces (pockets) for the nanoparticles are first created into the support, e.g. by partial perforation, followed by injecting the nanoparticles into said pockets.
• the nanoparticles injection can be performed by dispersing the nanoparticles in a fluid, optically transparent carrier (e.g. polymer or polymeric composition) and then injecting the resulting dispersion into the pre-formed support; preferably, the carrier is the same material making up the support, at the molten state; other examples of suitable carriers are the polymeric materials mentioned below in relation to layering.
• a fluid, optically transparent carrier e.g. poly
• the nanoparticles-containing and nanoparticles-free materials are then separately brought to a solid state (or gel-like state suitable for handling) and assembled together to form the support.
• the patterns can be obtained by printing, specifically by padprinting with silicone or similar stamps.
• the nanoparticles, suitably coated with PVP can be dispersed in acrylates and used for padding a pattern on a solid substrate, typically glass, plastics and the like. The viscosity of the dispersion determines the thickness of the pattern, in the range specified below.
• a procedure of uniformly dispersing the nanoparticles with the entire mass of the molten support prior to its casting in final shape is not contemplated: in fact, this would result in nanoparticles being present on the surface of the support coming into contact with the cell culture, which is to be avoided according to the invention.
• the layering is performed in correspondence of the relevant portions of the support, as previously defined. Layering is applied on the surface of the support opposite to the one on which the cell culture is to be held, i.e. beneath the support. Layering is preferably obtained by mixing the nanoparticles with a polymeric material (i.e. polymer or polymeric composition) and by laying down the resulting mixture on the support.
• a polymeric material i.e. polymer or polymeric composition
• the used polymer is optically transparent and can be indifferently chosen among those suitable as basis for layering: a non-limitative list thereof includes polyvinyl polymers, polysaccharides, polylactides, polyglycolides, polyacrylates, polymethacrylates, polycyanoacrylates, polyoleolefins, polyurethanes, polyamides, polyimides, polyethers, polyesters, polyacetates, polycarbonates, rubbers, polysiloxanes, cross-linked derivatives thereof and mixtures thereof.
• the polymer layer is made of polyvinyl alcohol, polyvinyl pyrrolidone and/or chitosan.
• the nanoparticle-containing polymer composition prior or after layering, is cross-linked.
• This contributes to immobilizing the nanoparticles, preventing their aggregation, release and/or leaking thus contributing to the efficiency and stability of thermal response of the support.
• cross-linking improves in general the support stability and resistance.
• the cross-linking can be obtained by adding to the polymer mixture an appropriate cross-linking agent, e.g. citric acid or other cross-linking agent selected depending on the specific chosen polymer. For example, non-chemical, physical cross-linking can be also applied.
• the layering can include the deposition of one layer or multiple layers, being equal or different from each other in composition.
• the thickness of the nanoparticle layer ranges preferably from 30 and 200 pm, preferably from 70 and 160 pm, more preferably from 80 and 120 pm, e.g. 100 or 110 pm.
• the manufacturing method will include a step of support hardening, normally performed by cooling. If the nanoparticles have been layered, the method also includes a step of layer hardening, normally obtained by cooling and/or, where applicable, curing the layer. Where possible, the hardening of support and layer may be combined in a single step, e.g. by cooling the nanoparticle-layered support at a suitable temperature.
• a further object of the invention is an incubator for biological samples, in particular cell cultures, including inside one or more supports as above described; in this embodiment, the supports are structural components of the incubator, i.e. they are mechanically, possibly permanently attached to the incubator; for this purpose, any suitable incubator as known in the art can be modified by mechanical integration of the supports of the present invention.
• the incubator is of industrial scale, containing inside a plurality of supports in accordance with the invention, each one with associated irradiating means, e.g. visible-red lamps (preferably LED), located in proximity of the support and focused thereupon. This disposition allows to perform a highly targeted stimulating thermal treatment of cell cultures, impossible to realize by traditional incubators not including the selectively heatable supports of the invention.
• a further object of the invention is a method of using the above defined support, obtaining cell differentiation with high efficiency, comprising: (i) placing a cell culture in contact with the support and (ii) irradiating the support with wavelength between 0.4 pm and 1.2 pm.
• An embodiment of this method, performed on neuronal cell cultures allows to obtain a highly efficient differentiation of the neuronal cells, i.e. obtaining high levels of neurite elongation and electrical activity in a limited incubation time.
• this can be obtained without the addition of differentiating chemical agents in the cell culture medium.
• step ii) said irradiating is performed until reaching a target temperature between 40.5 °C and 42.5 °C, preferably 41.5 °C, in the cell culture and maintaining said target temperature for 5 to 60 minutes, preferably 10 to 40 minutes, e.g. 30 minutes; then repeating the procedure (i-ii) for one or more times, e.g. two, three, four or five times, at interval of 24 h; in particular, repeating the procedure for one time was found suitable to reach the aims of the present invention and is preferred for reasons of economy/ simplicity; repeating the procedure for more than one time remains however possible.
• the supports used in the present methods allow to transfer heat to the cell culture in highly efficient way with high spatial (tens of microns; 35- 40 pm) and time resolution (seconds).
• the nanoparticles within the support provide heat sources being highly concentrated and highly focused towards the cell culture; moreover, the nanoparticle- containing support can produce a steeper temperature gradient, both upon warming and upon cooling, as compared to heating provided by bulk cell incubator.
• the focused irradiation is particularly effective when performed daily for 5 to 60 minutes, preferably 10 to 40 minutes, e.g. 30 minutes, at a temperature of 40.5 to 42.5, preferably 41.5°C.
• the invention is now described by way of the following non-limiting experimental examples.
• F-l l cells (mouse neuroblastoma N18TG-2 x rat DRG, ECACC Cat#08062601 RRID: CVCL_H605; Platika, Doros & Boulos, M & Baizer, L & Fishman, M. (1985).
• Biocompatible and FDA approved Prussian Blue nanoparticles exhibit intense absorption in the 700-750 nm region due to the metal-to-metal charge transfer between Fe II and Fe III through the formation of a cyanide bridge, and light irradiation of this band results in thermal relaxation.
• Prussian Blue nanoparticles were prepared according to literature (Dacarro G, Grisoli P, Borzenkov M, Milanese C, Fratini E, Ferraro G, Taglietti A & Pallavicini P. (2017). Self-assembled monolayers of Prussian blue nanoparticles with photo-thermal effect. Supramolecular Chemistr. 29: 11, 823-833) increasing from 1 mM to 10 mM the concentration of the reagents; 100 ml of a 10 mM FeCh solution were mixed with 10 mM K 4 [Fe(CN) 6 ] in 0.025 M citric acid and heated to 60 °C under stirring. After 1 min stirring at 60° C, the solution was cooled at room temperature.
• the solution was centrifuged for 25 min at 13,000 rpm in 10 ml test tubes for purification. Centrifuged Prussian Blue nanoparticle pellet was resuspended in half the original volume. The absorbance peak of PB aqueous solution was evaluated with Jasco, V- 570 spectrophotometer.
• a solution containing 7% of polyvinyl alcohol (PVA, average molecular weight 72000 g mol-1, degree of hydrolysis 98%, Sigma- Aldrich, St. Louis, MO, USA) and 27-30% of 10 mM Prussian Blue nanoparticles was made.
• the PVA powder was dissolved in water and was maintained in oven at 70°C for at least one hour. Then the PB solution was added and the mixture was achieved by an hour under continuous stirring.
• a Ti:Sa laser (Mai-Tai DeepSea Ti: Sapphire®, Spectra Physics®, Santa Clara, CA, USA) tunable between 0.69 pm and 1.10 pm was used to increase the temperature of the cells into the petri dish in correspondence of the PVA-PB layer. By exploiting the 0.72 pm wavelength, the power was chosen in order to reach the desired temperature. Temperature calibrations were made either on dry Petri dishes, in order to check the reproducibility of the layers, and on Petri dishes with 2 ml of culture medium, in order to determine the power for temperature increase. An accurate calibration was performed by a thermocamera (FLIR E40, FLIR Systems Inc., OR, USA) and by a hypodermic probe (Omega Engineering Ltd, Stamford, CT).
• the laser spot size has also been accurately calibrated in order to cover the whole layer area.
• a beam expander has been placed on the beam path and the spot size has been measured by recording the power after passing through a variable diameter iris. Fitting of the curve of power versus the iris diameter gives the spot at full size.
• the Petri dish was placed on the sample holder and the laser beam irradiated it from below. During the irradiation the Petri dishes were maintained within a 37°C home-made chamber whose temperature was controlled by The Cube (Life Imaging Services, Basel, CH).
• Morphology was determined by imaging the cells for 8 days from seeding. Cells were seeded on day 0, and on day 1 images were taken in transmitted light mode (see in the following); after a recovery of at least 2 hours, cells were irradiated for the first time. The irradiation was performed until reaching a target temperature of 41.5 °C in the cell culture and maintaining said target temperature for 30 minutes; during the rest of incubation time, the cells were maintained at a temperature of 37°C. The same procedure was repeated on day 2. Electrophysiological recordings were performed on days 7 and 8.
• Transmitted images were acquired on a Leica SP5 microscope (Leica Microsystems, Wetzlar, D) with an air objective (20X HCX PL Fluoter, Leica Microsystems, Wetzlar, D).
• Six tiles mode images were acquired in order to fully cover the irradiated area where the layer was present or an equivalent one for control and stimulated samples. In this way the same area was always imaged for the 8 days, and comparative statistical analysis was achieved among different experiments.
• the images were processed by ImageJ (Version 2.0.0, Opensource code). Neurites were manually traced, then a homemade macro that subtracted each traced image from the original one was run. The new image, on which only the traced neurites were visible, was then binary converted and skeletonized. The characteristics of the traced neurites were extracted in a text file and analyzed by the software Origin 9 (OriginPro, Version 2019, OriginLab Corporation, Northampton, MA, USA).
• LDH lactate-dehydrogenase
• the samples used for the assay were maintained at -20°C. According to the protocol, they were defrosted in ice, centrifuged at 1000 rpm for 4 minutes and the supernatant was collected for the assay. 1 ml (total volume) of solution was made, which contained (pi): K-phosphate Buffer, 850; NADH, 20, and stimulated or control samples, 70. The reaction started by adding 60 m ⁇ of Piruvate. The rate of the absorbance decrease over time was measured and the ratio of LDH activity (U/ml) into the cell culture medium was calculated by using the standard formula.
• the morphological characterization showed that samples stimulated according to the invention (Panel B) had a typical neuronal morphology and were able to form several small neuronal networks into the Petri dish; this was not evident from the controls (Panel A) .
• the graphs in Figure 2 showed that the irradiated cells according to the invention (IR) had significantly longer neurites compared to the control cell culture not thermally treated (CTRL) or treated with the same IR irradiation protocol but with no nanoparticles incorporated in the support (IR NOPB), these differences being particularly marked on days 7 and 8.
• Figure 2 also shows that cells grown on supports not containing PBNPs and irradiated had a trend comparable to the control on all days of the experiment; moreover, they had shorter neurites compared to irradiated samples with PBNPs, suggesting that the single NIR Laser did not induce a consistent differentiation in F- 11 cells.
• electrophysiological investigation was performed on irradiated cells to verify the eventual development of the typical properties of electrically mature neurons.
• the electrophysiological parameters investigated were the electrical activity, sodium and potassium current densities and the resting membrane potential. As shown in Figure 3, all the parameters tested were significantly changed in irradiated samples, even the sodium current density.
• Irradiated samples showed a percentage of cells with spontaneous activity significantly higher compared to 37°C cells (p ⁇ 0,001, Chi-Square Test), indicating that this method could induce F-l l cell line to differentiate.
• LDH lactate-dehydrogenase

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