Method for controlled cargo delivery to cells, device and use thereof

Abstract

The current invention relates to a method for applying cargo to a cell positioned on a substrate. This method involves providing a drop of a solution comprising a cargo onto a substrate where one or more cells are present. The drop is partially covering the cells. Following this, a force is applied to the drop, thereby spreading the solution into a thin layer covering the cells. Finally, the delivery of the cargo into or onto the cells is performed. Furthermore, the invention also relates to an assembly and use of the assembly with the described method.

Description

[0001] METHOD FOR CONTROLLED CARGO DELIVERY TO CELLS, DEVICE AND USE THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a method, device and use thereof in biotechnology, more specifically, it applies to controlled cargo delivery to cells.

[0004] BACKGROUND

[0005] Efficient and controlled delivery of cargo to cells is essential in various fields, including molecular biology, biomedical diagnostics, gene therapy, and drug discovery. Existing transfection and cargo delivery methods, however, present several limitations that affect reproducibility, efficiency, and resource consumption. Many conventional techniques rely on bulk liquid handling, in which the liquid volume is substantially larger than the volume of cells, leading to a high consumption of cargo.

[0006] When it comes to the intracellular delivery of cargo into cells, often referred to as transfection, a major challenge lies in achieving high transfection efficiency while maintaining adequate cell viability. Techniques such as lipofection or viral vectors can be effective in some contexts, but they can also introduce cytotoxic side effects or other complications. Physical techniques, including photoporation and electroporation, circumvent certain drawbacks by using physical energy to open pores in cell membranes. However, while effective, these methods can suffer from diffusion limitations, non-optimal distribution of cargo near the cellular membrane, and overall inefficiencies if the cargo is present in low concentration or is incompletely contacting the cell surface.

[0007] Another background consideration is the environment in which cargo delivery onto or into cells takes place. Often, cells are cultured in multi-well plates or other compartmentalized substrates in which the volume of cell culture medium is substantially larger than the total volume of cells. Diluting the cargo in such large volumes of liquid hamper efficient delivery. Moreover, reducing the liquid volume can result in inconsistent contact between the cargo-laden medium and the cells' surfaces, where fluid volumes can pool, menisci can form, and uniform coverage may be difficult to achieve.To address these issues, there is increasing interest in "small volume" approaches, wherein the volume of solution over the cells is minimized or otherwise controlled. By reducing the liquid volume relative to the cell volume, the required amount of cargo can be reduced for the same delivery efficiency. Similarly, if the amount of cargo is kept the same, it will be present at a higher concentration, thus improving the delivery efficiency.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention and embodiments thereof serve to provide a solution to one or more of the above-mentioned disadvantages. To this end, the present invention relates to a method for applying cargo to a cell positioned on a substrate according to claim 1. Disclosed herein is a method for applying cargo to a cell positioned on a substrate, said method comprising:

[0010] (a) providing a drop of a solution comprising a cargo onto a substrate containing one or more cells;

[0011] (b) applying a force to said drop, thereby spreading said solution into a thin layer covering said cells; and

[0012] (c) delivering of said cargo into or onto said cells.

[0013] Further preferred embodiments are described in claims 2 to 12.

[0014] The present invention provides an improved method and assembly for delivering cargo to cells, addressing key limitations of the prior art by ensuring controlled, uniform, and efficient cargo distribution while minimizing reagent / cargo waste or expenditure.

[0015] This approach offers several advantages over existing methods. By using a precisely controlled spreading mechanism, it ensures uniform exposure of the cargo to all cells, improving reproducibility and reducing variability in transfection efficiency. The method enables the use of highly concentrated cargo solutions while limiting the total volume required, significantly reducing reagent consumption and associated costs.

[0016] In a second aspect, the invention relates to an assembly according to claim 13. Said assembly comprises a cell culturing device and an insert, wherein said cell culturing device comprises one or more compartments configured for containing one or more cells and a solution comprising a cargo to be delivered into or onto said cells, andwherein said insert is configured to fit within at least one of said compartments. A further preferred embodiment is shown in claim 14.

[0017] In a third aspect, the invention relates to the use of said assembly according to claim 15.

[0018] DESCRIPTION OF FIGURES

[0019] Figure 1 is a schematic overview of a transfection process according to an embodiment of the current invention.

[0020] Figure 2 presents the efficiency of mRNA transfection according to the experiment as performed in Example 1.

[0021] Figure 3 shows the fold reduction in mRNA consumption achieved according to the experiment as performed in Example 1.

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] In a first aspect, the current invention relates to a method for applying cargo to a cell positioned on a substrate. This method involves step (a) of providing a drop of a solution, such as an aqueous solution, comprising a cargo onto a substrate where one or more cells are present. Due to surface tension, the drop may only be partially covering the cells. Following this, according to step (b) a force is applied to the drop, thereby spreading the solution into a thin layer covering the cells. Finally, according to step (c) the delivery of the cargo into or onto the cells is achieved.

[0024] Cell transfection is a critical process in molecular biology, used to introduce genetic material, proteins, or other biomolecules into cells. In addition to introducing such materials into cells, this method can also facilitate the delivery of molecules or compounds, such as dyes, markers, or antibodies, onto the cell surface, such as for labeling or diagnostic purposes. Current methods often require large volumes of reagents, materials, molecules and / or compounds, making them costly and resource-intensive.

[0025] In the context of this invention, the term "cargo" refers to any substance, molecule or particle intended for delivery into or onto a cell.The current invention addresses these challenges by introducing a method that minimizes resource usage while maintaining or enhancing the delivery efficiency. By using a small droplet of the cargo solution, this approach ensures that only the necessary amount of material is used, reducing waste and associated costs. The application of a force, preferably performed by contacting the drop with an insert, to spread the droplet ensures uniform coverage, allowing for consistent interaction between the cells and the solution. This innovation is particularly relevant for research and industrial laboratories that aim to achieve reproducible results with minimal reagent consumption.

[0026] Delivery of the cargo onto or into the cells is achieved by diffusion and / or flow of the cargo to the cell surface, optionally followed by internalization into the cells through various mechanisms, such as endocytosis, passive diffusion, facilitated diffusion or active transport. Endocytosis, the process by which cells internalize molecules by engulfing them, encompasses different types, such as phagocytosis, pinocytosis, macropinocytosis, receptor-mediated endocytosis, clathrin-mediated endocytosis, caveolin-mediated endocytosis, flotillin-mediated endocytosis, Arf6 mediated endocytosis, CLIC / GEEC dependent endocytosis. Intracellular delivery can also happen through membrane fusion, pore formation, or lipid bilayer disruption. Pore formation or lipid bilayer disruption may be caused by the cargo itself or by an additional treatment before or after applying the insert. Non-limitative exemplary treatments are chemical permeabilization, electroporation, mechanoporation, sonoporation, photoporation, optoporation, magnetofection, ballistic delivery, or puncturing with micro- or nanoneedles.

[0027] In an embodiment a force is used to spread the solution into a thin layer across the substrate, ensuring that the cargo is evenly distributed and comes into direct contact with the cells. The spreading mechanism enhances the interaction between the cells and the cargo, increasing the likelihood of successful delivery. This uniform contact is particularly important for achieving consistent and reproducible results, as uneven distribution can lead to variable transfection efficiencies, or uneven staining or labelling, and hinder downstream applications.

[0028] This approach to cargo delivery is advantageous because it allows the use of highly concentrated solutions in small volumes. Traditional methods often rely on larger quantities of diluted solutions, which can reduce the effectiveness of the cargo and increase the overall cost. By concentrating the cargo within a small volume, this method maximizes the efficiency of the delivery process while minimizing theconsumption of expensive reagents. Furthermore, the controlled uniform spreading process reduces waste and ensures that the cargo is applied evenly, further contributing to its efficiency and consistency.

[0029] In addition to improving efficiency and cost-effectiveness, the method enhances the versatility of cargo delivery. It is compatible with various cell types, substrates, and cargo formulations, making it suitable for diverse applications in basic research, drug discovery, biomedical diagnostics and therapeutic development. Whether used for the binding of ligands to cell surface receptors, delivering genetic material to study gene expression, introducing proteins for functional assays, or staining or labelling cells, this method provides a reliable and scalable solution for achieving efficient cargo delivery with minimal resource consumption.

[0030] In an embodiment of step (a), the a solution, optionally an aqueous solution, with cargo is provided or deposited on said substrate with cells by conventional means known in the art such as pipetting or spraying. The preparation of the sample prior to deposition is needed for ensuring efficient and reproducible cargo delivery. This preparation includes selecting appropriate cargo formulations, optionally mixing the cargo with suitable transfection aids, and optimizing the solution conditions to enhance uptake efficiency.

[0031] The cargo, which may include viruses, viral-like particles, nucleic acids, proteins, nanoparticles, antibodies, other biomolecules, dyes, markers, drug molecules, or combinations thereof - further examples are described below -, can be prepared in a solution, such as an aqueous solution, to maintain stability and bioactivity. Common systems include phosphate-buffered saline (PBS), Tris-HCI, HEPES buffer, or optically clear media such as DMEM or RPMI when working with live cells. The pH and ionic strength of the solution can be controlled to maintain high cell viability while avoiding aggregation or degradation of the cargo. For instance, nucleic acids may require the presence of magnesium or calcium ions for stability, whereas proteins may require carrier molecules such as bovine serum albumin (BSA) to prevent adsorption to plastic surfaces.

[0032] For enhanced transfection efficiency, the cargo may be mixed with transfection aids or delivery vehicles prior to application. This includes the use of lipid-based carriers such as liposomes or lipid nanoparticles (LNPs), which encapsulate nucleic acids or proteins and facilitate cellular uptake via endocytosis. Alternatively, polymeric transfection reagents, such as polyethylenimine (PEI) or dendrimers, can be used toform cargo-polymer complexes that improve stability and cellular entry. Electrostatically driven complexes, such as cationic lipids binding to negatively charged DNA or RIMA, are often used for gene delivery applications.

[0033] In some applications, the preparation may involve the use of stimuli-responsive particles to enable intracellular delivery and / or cargo release with the aid of an external stimulus, such as light, mechanical pressure, electricity or magnetism. In some embodiments, these particles are pre-loaded with molecules of interest, such as nucleic acids. In other embodiments, these particles are mixed together with other molecules of interest in the solution without being conjugated to one another. For example, if the method incorporates magnetofection, magnetic nanoparticles loaded with molecules of interest are dispersed in the solution and guided towards the cells using an external magnetic field after deposition. In another example, if the method incorporates photoporation, photothermal nanoparticles are mixed with molecules of interest and added to the cells. Next, a light trigger is applied to permeabilize the cell membrane through photothermal effects, allowing the molecules of interest to migrate into the cells.

[0034] For high-throughput applications, the solution containing the cargo can be dispensed using automated liquid handling systems, ensuring precise and consistent droplet placement across multiple wells or culture chambers.

[0035] Prior to cell seeding, the substrate with adherent or resting cells should be prepared to optimize cargo interaction. This may include coating the substrate with extracellular matrix proteins such as fibronectin, laminin, or collagen to promote cell attachment and viability. Additionally, for certain experimental conditions, the cells may be pre-conditioned with media supplements or serum-free formulations to improve delivery efficiency while minimizing cytotoxicity.

[0036] By carefully preparing the solution and optimizing the sample conditions before deposition, this embodiment ensures consistent cargo delivery, high delivery efficiency, and minimal variability across different experimental setups.

[0037] In an embodiment, step (b) of the method is performed by contacting the drop with an insert. The insert interacts with the solution, causing it to spread across the substrate and form a thin, preferably uniform, layer over the cells. By ensuring even distribution, this step enhances the uniform interaction between the cargo and the cells, optimizing the conditions for uptake.The use of an insert for spreading the drop offers several advantages over alternative methods such as pipetting, shaking, or tilting the substrate. It provides a reproducible and controlled means of spreading, minimizing variability between experiments. The insert can be designed with specific material properties or surface coatings to influence how the solution interacts with the substrate and the cells. Depending on the cargo and cellular requirements, variations in the insert's shape, texture, or rigidity can further refine the delivery process. This approach ensures consistency in delivery efficiency while reducing the risk of uneven fluid distribution that might otherwise lead to uneven exposure or unintended loss of cargo.

[0038] In an embodiment, step (b) of the method is performed by moving and / or pressing at least part of the insert onto the drop and / or towards the cells on the substrate. This ensures a substantially homogeneous spreading of the droplet, forming a thin fluid layer between the insert and substrate surface. The controlled motion and pressure applied by the insert ensure that the solution is evenly distributed over the cells, promoting efficient contact between the cargo and the cell surface.

[0039] The movement of the insert can be executed in various ways, such as a linear pressing motion, a rotational spreading motion, or a combination of both. The movement can be performed manually or by using automation. This versatility allows for customization of the method to suit different types of substrates, cell arrangements, and experimental setups. The pressing force can be calibrated to ensure that the cells remain intact and viable while still achieving an effective spreading of the solution. For sensitive cell types, the applied force may be gentle and distributed evenly across the insert to minimize stress on the cellular structure.

[0040] By transforming the droplet into a thin layer, the method maximizes the surface area of the solution in contact with the cells, enhancing the delivery efficiency of the cargo. The planar shape of the solution ensures uniform coverage, reducing variability in cargo delivery across different regions of the substrate. At the same time it ensures coverage of cells by the fluid, as is typically required to maintain cell health.

[0041] In an embodiment, the cells are adherent to or are resting on said substrate. Adherent cells, such as fibroblasts, epithelial cells, or endothelial cells, may naturally attach to a substrate, providing a fixed position that facilitates interaction with the applied solution. This adherence allows for consistent and uniform contact with the cargo-containing solution, as it minimizes cell displacement during the spreadingprocess, thereby enhancing the efficiency and reproducibility of the delivery process. Furthermore, adherent or resting cells provide a controlled environment for studying the effects of the cargo, enabling downstream analysis with minimal variability due to cell movement or uneven exposure.

[0042] For cells that are not adherent, it may be beneficial to enhance their adhesion to the substrate in order to prevent them from displacing when the insert is applied. To enhance adhesion of non-adherent cells, preparation techniques can be used such as coating the substrate with cell-adhesion-promoting molecules (e.g., poly-L-lysine, fibronectin, or collagen) or by using specialized cell culture materials that promote temporary adherence. In another embodiment, cells may be present at the surface or bottom of a recipient, but without being adherent to the latter. For instance, said cells may be present in pellets, after centrifugation.

[0043] The embodiment where cells are adherent to or are resting on said substrate is particularly useful for applications involving delicate cellular structures or processes, such as neuronal cultures or differentiated stem cells, where maintaining the spatial integrity of the cells is critical for achieving desired outcomes. By ensuring that the cells are adherent to or resting on the substrate, the method achieves both efficiency in cargo delivery and consistency across experimental replicates.

[0044] In an embodiment, the cells used in this method can be chosen from the group of, but not limited to, immune cells such as T-cells, epithelial cells, fibroblasts, endothelial cells, hepatocytes, mesenchymal stem cells, induced pluripotent stem cells, hematopoietic stem cells, osteoblasts, myocytes, neuronal cells, adipocytes, chondrocytes, keratinocytes, smooth muscle cells, cardiac muscle cells, pancreatic beta cells, astrocytes, oligodendrocytes, Schwann cells, retinal pigment epithelial cells, trophoblasts, melanocytes, glial cells, corneal epithelial cells, alveolar epithelial cells, podocytes, vascular smooth muscle cells, tenocytes, and synoviocytes, as well as prokaryotic cells such as but not limited to Escherichia coli, Bacillus subtilis, and Streptomyces spp., plant cells such as but not limited to mesophyll cells, guard cells, root hair cells, and tracheary elements, and insect cells such as Sf9 and S2 cells.

[0045] The volume V of the drop to be applied depends on the substrate surface area S, and can be calculated according to the formula V = h*S, where h is the thin layer thickness.After spreading the droplet into a thin layer, the layer thickness may for instance be between 50-300 pm (further preferred ranges below), with the reduction factor depending on factors such as the volume of the droplet, the hydrophilicity of the substrate and insert, and the characteristics of the insert or spreading tool used.

[0046] A minimum thickness of 50 pm allows for complete coverage of the cells while preventing gaps in the solution layer, ensuring consistent exposure to the cargo.

[0047] The upper limit of 300 pm accommodates situations where slightly thicker layers are necessary, such as for substrates with uneven surfaces, higher cell densities or larger cells or cell clusters.

[0048] However, the preferred range of 50 to 300 pm is particularly beneficial for conserving resources by using minimal volumes of the solution containing cargo, making the method both cost-effective and environmentally friendly. Furthermore, the controlled thickness of the layer ensures reproducibility across experiments, as the cargo is distributed in a consistent and predictable manner, reducing the volume of the solution with cargo, hence minimizing the required amount of cargo, and enhancing the efficiency of the delivery process. Moreover, the preferred range also allows placing and removing the insert without causing substantial damage to the cells.

[0049] In an embodiment, the thin layer has a thickness of between 1 pm and 1 mm, preferably of between 2 and 500 pm, preferably of between 50 and 300 pm, more preferably between 50 and 150 pm. This thickness range ensures that the aqueous solution is distributed uniformly over the cells, providing optimal contact between the cargo and the cellular surface for efficient delivery or labeling / staining.

[0050] In an embodiment, the drop has a volume between 0.5 and 250 pL. A minimum volume of 0.5 pL ensures that there is sufficient solution to interact with the cells and spread into a thin, uniform layer, while the maximum volume of 250 pL prevents excessive overflow or overconsumption of cargo.

[0051] In preferred embodiments, the drop has a volume of between 1 and 250 pL, preferably between 1 and 200 pL, preferably between 1 and 150 pL, preferably between 2 and 150 pL, preferably between 3 and 150 pL, or between 1 and 100 pL, or between 2 and 90 pL, or between 3 and 80 pL, or between 4 and 70 pL, or between 5 and 60 pL, or between 5 and 50 pL. This range of volumes allows for precisedelivery of the solution while accommodating a variety of experimental setups and substrate sizes.

[0052] A preferred range of between 3 and 150 pL provides an optimal balance between achieving uniform coverage of the cells and maintaining a high cargo concentration. Smaller volumes within this range are particularly advantageous for high-value cargo or resource-limited applications, where minimizing waste is a priority. Larger volumes within the range may be better suited for experiments involving larger substrates or higher cell densities, ensuring complete coverage without compromising the concentration of the cargo.

[0053] This volume range also enhances the flexibility of the method by making it adaptable to various experimental conditions. For instance, smaller volumes are ideal for microfluidic applications or small-scale experiments, while larger volumes can accommodate multi-well plates or larger culture dishes. The ability to precisely control the droplet volume ensures consistency in cargo delivery and contributes to the reproducibility of the method, which is particularly important in both research and industrial settings. Finally, the volume should preferably be sufficient to fully or at least sufficiently cover the cells upon spreading while maintaining a controlled and uniform thin layer.

[0054] The drop volume may also depend on the substrate size and / or form. In practice, in view of the widespread use of 96-, 24-, and 6-well plates, a preferred volume is generally between 3 and 150 pL. As non-limiting examples, for cells in a 96 well plate, the droplet volume may range from 3-9 pL. After spreading the droplet into a thin layer, the layer thickness may be between 100-300 pm. For cells in a 24 well plate, the droplet volume may range from 10-30 pL, with a resulting layer thickness of 50-150 pm. For cells in a 6 well plate, the droplet volume may range from 50-150 pL, with a resulting layer thickness of 50-150 pm.

[0055] In an embodiment, said thin layer will cover at least 95%, more preferably 96%, more preferably 97%, more preferably 98%, more preferably 99% up to 100% of said cell substrate and / or cells on said cell substrate.

[0056] In an embodiment, step (c) of the method, intracellular delivery of the cargo, is facilitated using an intracellular delivery method. This intracellular delivery method comprises or can be photoporation, electroporation, lipofection, polyfection, sonoporation, magnetofection, optoporation, laser-assisted transfection,mechanoporation, viral transduction or combinations thereof. Each of these techniques leverages distinct mechanisms to enhance the uptake of the cargo by the cells, offering flexibility to tailor the method for various experimental conditions, cell types, and cargo.

[0057] In a preferred embodiment, step (c) of the method is performed using photoporation with a pulsed laser and photo-responsive particles, such as photothermal nanosentizers.

[0058] Lipofection and polyfection employ lipid- and polymer-based reagents to encapsulate the cargo, facilitating its fusion with the cell membrane and subsequent delivery into the cell. This gentle technique is suitable for sensitive cell types and is especially effective for transfection with DNA or RIMA. Sonoporation uses ultrasound waves to generate microbubbles that create temporary openings in the cell membrane, promoting cargo uptake. This method is advantageous for its ability to target large populations of cells simultaneously.

[0059] Magnetofection utilizes magnetic fields to guide magnetic particles bound to the cargo towards the cells, enhancing delivery efficiency. This approach is particularly useful for localized applications or when working with substrates that can be influenced by magnetic fields. Optoporation and laser-assisted transfection employ advanced optical techniques, such as focused laser beams, to transiently permeabilize the cell membrane with high precision, making them ideal for applications requiring targeted delivery.

[0060] Photoporation involves the use of light, often from lasers, to create transient pores in combination with photothermal nanosensitizers in the cell membrane, allowing the cargo to enter the cell. This technique is particularly precise and can be localized to specific areas of the cell culture, minimizing off-target effects.

[0061] Electroporation, on the other hand, uses short, high-voltage electric pulses to temporarily disrupt the cell membrane, enabling the cargo to pass into the cell. This method is highly effective for a broad range of cell types and is widely used for delivering nucleic acids and other biomolecules.

[0062] Mechanoporation uses a mechanical force to permeabilize the outer cell membrane, for instance using fluid flows or physical objects that pose shear stress on the cell membrane. Viral transduction uses viral vectors to transduce cells.In an embodiment, these techniques can also be combined to optimize cargo delivery based on specific needs. For example, lipofection may be paired with electroporation to enhance the efficiency of nucleic acid delivery, or sonoporation could be combined with magnetofection for targeted delivery of nanoparticles. The choice of method or combination depends on factors such as the type of cargo, the physical and biological characteristics of the cells, and the intended application. This flexibility ensures that the method can be customized to achieve optimal results across a wide range of experimental and therapeutic scenarios.

[0063] In a preferred embodiment, photoporation is used.

[0064] In an embodiment, the solution comprises one or more transfection aids, wherein said transfection aids are selected from physical, chemical or biological agents that enhance cellular uptake of biomolecules. Preferably, the transfection aids comprise nanoparticles, liposomes, cationic polymers. Even more preferably, the transfection aids comprise photo responsive particles, which are particles that change their properties or behavior in response to light exposure. These particles can be activated by specific wavelengths of light, allowing for precise spatial and temporal control over their interaction with cells. When incorporated into the solution, photo-responsive particles can facilitate or enhance the delivery of the cargo to the cells by leveraging light-induced mechanisms such as localized heating, membrane disruption, or controlled release of the cargo.

[0065] The use of photo-responsive particles enables non-invasive activation, making the method particularly suitable for sensitive or delicate cell cultures. The particles can be engineered to respond to light of specific wavelengths, allowing for multi-level control over the cargo delivery process. For instance, particles responsive to different wavelengths can be included in the same solution, enabling sequential activation of distinct cargos in a single experiment.

[0066] In an embodiment, the photo-responsive particles may comprise nanobombs, preferably multiple nanobombs. The at least one nanobomb comprises n first particles and m second particles, with each of n and m being at least one. At least one of the m second particles is in close proximity to at least one of the n first particle. Preferably, a first particle of a nanobomb is surrounded by one and more preferably by more than one second particle in close proximity to this first particle. The first particle that is surrounded by the one or more second particles is therebyreferred to as the central first particle. The second particle or second particles surrounding such first particle is / are referred to as the surrounding particle(s).

[0067] The first particles are able to absorb electromagnetic radiation such as to generate a vapor bubble, for example a vapor microbubble or vapor nanobubble, whereby the generation of the vapor bubble causes the at least one second particle (i.e. the at least one of the m second particles that is in close proximity to at least one of the n first particles) to be propelled over a distance D away from said at least one first particle, with said distance D being larger 0.01 pm. The distance D being defined as the displacement of the at least one second particle as a consequence of the process of generating vapor bubbles.

[0068] Preferred first particles comprise a metal, a metal oxide, carbon or a carbon based material, a light absorbing material or a material loaded or functionalized with one or more light absorbing material or compound(s) or a combination thereof. First particles may for example comprise a combination of metals, a combination of metal oxides, a combination of carbon based materials, a combination of light absorbing materials or a combination of materials loaded or functionalized with one or more light absorbing material or compounds or any other combination.

[0069] Examples of metals comprise gold, silver, platinum, palladium, copper and alloys thereof. Preferred metals comprise gold, silver and alloys thereof.

[0070] Examples of metal oxides comprise iron oxide, titanium dioxide, zirconium oxide, cerium oxide, zinc oxide and magnesium oxide, and further also metal oxides incorporating more than one metal such as spinel (MgAhCM), perovskite oxides (CaTiOs), lithium cobalt oxide (LiCoC ), and bismuth vanadate (BiVCM).

[0071] Examples of carbon or a carbon based material comprise graphene or graphene oxide.

[0072] Examples of a light absorbing compound comprises synthetic organic or inorganic absorbers as well as naturally occurring absorbers or derivatives thereof, for example light absorbing dye molecules such as indocyanine green, trypan blue, cyanine, and rhodamine, inorganic quantum dots (having low fluorescence quantum yield), naturally occurring light absorbers like pigments (such as melanin, rhodopsin, photopsins, iodopsin, chlorophyll, carotenoids, retinal, hemoglobin, porphyrins,flavins, and more) and synthetic analogs like polydopamine, or photosensitizers used in photodynamic therapy.

[0073] Preferred second particles comprise a polymer, a metal oxide, silicon or silicon oxide, liposome or liposomes or combinations thereof.

[0074] Preferred second particles comprise polymer particles (micro or nanoparticles), metal oxide particles (micro or nanoparticles), silicon or silicon oxide particles (micro or nanoparticles), liposomes, drug loaded polymer particles, drug loaded metal oxide particles, drug loaded silicon or silicon oxide particles and drug loaded liposomes.

[0075] Examples of polymer particles comprise polystyrene beads or poly(lactic-co-glycolic acid) (PLGA) beads.

[0076] Examples of metal oxide particles comprise iron, titania or zirconia microparticles or nanoparticles, and further also metal oxides incorporating more than one metal such as spinel (MgAhCM), perovskite oxides (CaTiOs), lithium cobalt oxide (LiCoO2), and bismuth vanadate (BiVCM).

[0077] In an embodiment, the photo-responsive particles may comprise organic photo-responsive particles, wherein said photo-responsive particle is selected from the group consisting of a polymer-based particle, a protein-based particle, a lipid-based particle, e.g. a liposome or a solid lipid particle, and / or combinations thereof.

[0078] Protein-based particles include protein-based nanoparticles (PNPs) such as non-viral particles based on ferritin, heat shock proteins (Hsp), DNA-binding proteins from starved cells (Dps), encapsulin, the E2 protein of pyruvate dehydrogenase, lumazine synthase, vault proteins; and virus-like particles (VLPs).

[0079] In embodiments, the protein may be an albumin such as human serum albumin (HSA). For example, human serum albumin ICG nanoparticles (HSA-ICG NPs) may be prepared as described in Sheng et al., 2014, ACS Nano, 8(12): 12310-22.

[0080] The lipid-based particles may be liposomes, solid lipid particles, extracellular vesicles, exosomes, lipid droplets, lipoproteins, micelles, and more, preferably liposomes or solid lipid particles.

[0081] In embodiments, the lipid-based particles may comprise a natural, a bacterial or a synthetic lipid. In embodiments, the lipid-based particles may comprise an anionic lipid, neutral lipid, cationic lipid, ionizable lipid or a sterol.Suitable examples of anionic lipids include phosphatidylserine (PS) and phosphatidylglycerol (PG).

[0082] Suitable examples of neutral lipids include prostaglandins, eicosanoids, glycerides, glycosylated diacyl glycerols, oxygenated fatty acids, very long chain fatty acids (VLCFA), palmitic acid esters of hydroxystearic acid (PAHSA), N-acylglycine (NAGIy), and prenols.

[0083] Suitable examples of cationic lipids include multivalent cationic lipids; 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA); ethylphosphocholines (EPC); dimethyldioctadecylammonium (DDAB); Nl-[2-((lS)-l-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); pH sensitive lipids; l,2-dioleoyl-3-trimethylammonium propane (DOTAP); 30-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Cholesterol); N4-Cholesteryl-spermine (GL67); l,2-dioleyloxy-3-dimethylaminopropane (DODMA); Dlin-MC3-DMA (MC3); DLinDAP; DLinDMA; DLinKDMA; or DLinKC2-DMA.

[0084] In an embodiment, the photo-responsive particle cannot be coupled or be coupled to and / or embedded in a solid support or solid material, such as a porous or a non-porous structure, preferably a porous structure. In a preferred embodiment, the photo-responsive particle is coupled to and / or embedded in a solid support or solid material. Examples comprise a porous polymer structure with the particles embedded in the porous polymer structure.

[0085] Examples of porous structures comprise structures comprising fibers (for example polymer fibers), structures comprising particulates (for example polymer particulates), structures comprising a combination of fibers and particulates (for example a combination of polymer fibers and / or polymer particulates) and structures comprising foam (for example polymer foam). The fibers and / or particulates can be interconnected or not. The particulates as for example the polymer particulates may comprises spherical particulates as well as irregular shaped particulates. The particles able to absorb electromagnetic radiation are preferably embedded in the fibers, the particulates, or the foam, preferably in such a way that the particles are not (partially) exposed to the free area surface of the structure.

[0086] Preferred examples of polymer comprise polystyrene fibers, polycaprolacton fibers, ethylcellulose fibers, cellulose acetophthalate fibers, polylactic acid fibers and polylactic-co-glycolic acid based fibers. The (polymer) fibers can be surface modified.Preferred examples of particulates comprise polystyrene, polycaprolacton, ethylcellulose, cellulose acetophthalate, polylactic acid, polylactic-co-glycolic acid based fibers. The polymer particulates can be surface modified.

[0087] In an embodiment, the insert comprises a substantially flat surface as a base, which is contacted with the drop of the solution. The flat surface ensures uniform application of force across the droplet, allowing the solution to transform from its initial spherical shape into a thin, even layer that effectively covers the cells. By maintaining a smooth and consistent contact area, the insert promotes reliable distribution of the cargo, minimizing variability in delivery efficiency across different regions of the substrate.

[0088] In an embodiment, the base of the insert can have various shapes to accommodate different applications and well geometries. It may be disc-shaped, rectangular, square or oval, depending on the design of the cell culturing device. The shape can be tailored to optimize coverage while maintaining reproducibility and efficiency in cargo delivery.

[0089] Attached to the center of the base, the insert may include a vertical handle or protrusion that facilitates handling and precise positioning of the insert onto the droplet. This handle can be designed with a cylindrical, conical or other ergonomic shape to accommodate manual or automated use.

[0090] The size and proportions of the base can vary depending on the size of the substrate and the area of cells being targeted. For instance, the base may have a diameter that allows it to effectively cover the droplet while avoiding contact with areas outside the intended substrate region. The handle or protrusion can also vary in height and width, ensuring stability during use and compatibility with different application techniques.

[0091] In an embodiment, the base will have a surface area that corresponds to at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably 100% of a corresponding surface with cells.

[0092] In an embodiment, the diameter of the base corresponds to at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least98%, more preferably at least 99%, more preferably 100% of the corresponding diameter of the surface with cells.

[0093] In an embodiment, the insert may be constructed from materials such as plastic (such as, but not limited to polystyrene (PS), polypropylene (PP), Polyethylene terephthalate glycol (PETG), polyvinylchloride (PVC), polycarbonate (PC)), glass or metal, with properties selected for compatibility with the solution and sterility requirements. Variations in design, such as a hollow or solid handle, may further enhance the versatility and functionality of the insert for different experimental conditions.

[0094] In a further embodiment, the insert can be designed to include a coating on its surface to enhance its functionality during the spreading of the solution. This coating can serve various purposes, such as reducing friction, preventing the adhesion of the solution to the insert, or ensuring compatibility with the cargo and the cells. The selection of a coating depends on the specific requirements of the method, including the type of cargo being delivered and the sensitivity of the cells. The coating may also play a role in maintaining sterility, ensuring biocompatibility, or preventing contamination.

[0095] The coating can be applied to the flat base, the handle, or both, depending on the desired properties. For instance, a hydrophilic coating may enhance the spreading of the solution by promoting uniform distribution across the cells. Conversely, a hydrophobic coating could be used to prevent unwanted retention of the solution on the insert. Antimicrobial or anti-biofouling coatings can be used to maintain sterility during repetitive use, while electrically conductive coatings may be suitable for applications requiring electric stimulation.

[0096] In an embodiment, the coating for the insert can be chosen from the group of, but not limited to, polytetrafluoroethylene (PTFE), silicone, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, parylene, gold, silver, titanium oxide, silica, hydrogels, carbon nanotubes, graphene oxide, antimicrobial peptides, alginate, cellulose derivatives, poly-L-lysine, polyethyleneimine (PEI), collagen, chitosan, Bovine serum albumin (BSA), plasma-deposited films, ceramics, epoxy resins, fluoropolymers, superhydrophobic nanostructures, and UV-curable coatings. Each of these coatings can be selected and tailored based on the specific requirements of the cargo delivery method and the experimental or industrial conditions.In an embodiment, the cells adhere to or rest on a substantially flat surface, which is provided with a texturized pattern. This patterned surface can influence various aspects of the method, including the spreading dynamics of the solution, the distribution of the cargo, and the interaction of the cells with the substrate. By incorporating a structured pattern, the surface can be engineered to optimize cell adhesion, enhance solution uniformity, and improve reproducibility in cargo delivery.

[0097] In an embodiment, the texturized pattern may include microstructures, nanostructures, or a combination of both. These features can be designed to control the wetting behavior of the solution, ensuring that the droplet spreads in a predictable and controlled manner. For example, a surface with microgrooves or ridges can guide the solution in a specific direction, promoting uniform coverage of the cells. Similarly, nanopatterned surfaces can modulate the interactions between the cargo and the substrate, influencing the stability of the thin layer after spreading.

[0098] In addition to enhancing the efficiency of cargo delivery, a texturized pattern can help manage the evaporation dynamics of the thin layer. By modifying the surface topology, the rate at which the solution evaporates can be adjusted, ensuring that the cargo remains in contact with the cells for an optimal period. This is particularly relevant for cargo types that require prolonged exposure for effective uptake, such as nucleic acids in transfection assays.

[0099] The patterning of the surface may be achieved through various fabrication techniques, including lithography, laser etching, embossing, plasma treatment, polymer casting, or combinations thereof. The choice of pattern design and fabrication method can be tailored to specific experimental needs, allowing for customization based on the type of cells, cargo, and substrate material. This embodiment provides an additional level of control over the transfection process, enabling fine-tuning of the method for increased efficiency, consistency, and adaptability across different applications.

[0100] In an embodiment, the cargo comprises a substance or agent intended for delivery into or onto a cell, such as a chemical, biochemical or biological substance. Such cargo can comprise, but is not limited to, DNA, plasmid DNA, cDNA, RIMA, mRNA, IncRNA, siRNA, miRNA, CRISPR-Cas9 complexes, plasmids, viral vectors, proteins, peptides, enzymes, antibodies, nanobodies, lipids, small molecules, dyes, fluorescent or luminescent markers / agents, chemotherapeutics, imaging labels e.g. for MRI or CT scanning (such as but not limited to zirconium oxide, and gadoliniumchelates or particles) nanoparticles, liposomes, exosomes, polysaccharides, synthetic oligonucleotides, transcription factors, cytokines, growth factors, immunomodulators, aptamers, molecular inhibitors, RIMA interference molecules, ribozymes, biosensors, synthetic transcription activators, chemical probes, therapeutic drugs, vaccine components, cell-penetrating peptides, hydrogels, magnetic particles, quantum dots, gold nanoparticles, polymeric nanoparticles, lipid nanoparticles, organelle-targeting probes, fluorescent nucleotides, metabolic tracers, and epigenetic modulators, viral-like nanoparticles, extracellular vesicles, photothermal nanoparticles, organic particles or drug loaded nanoparticles. In a preferred embodiment, the cargo comprises nucleic acids, oligonucleotides, ribozymes, proteins, polypeptides, peptides, peptidomimetics, peptide nucleic acids, antibodies, antibody fragments, antibody-like protein scaffolds, aptamers, photoaptamers, spiegelmers, chemical substances, lipids, carbohydrates, polysaccharides, viral vectors, viral-like nanoparticles, extracellular vesicles, photothermal nanoparticles, organic particles, drug loaded nanoparticles, or combinations thereof. The composition of the solution is tailored to the specific cargo, ensuring it meets the requirements for stability, activity and compatibility with the delivery method.

[0101] These cargo types are commonly used in a variety of biological and biomedical applications, including gene editing, therapeutic protein delivery, and studying intracellular processes. The method is versatile and can be tailored to different types of cargo, depending on the specific needs of the user or the application.

[0102] In an embodiment, the cargo has a concentration in the solution of between 1 pM and 1 M, preferably between 1 nM and 1 mM, more preferably between 10 nM and 100 pM, and most preferably between 100 nM and 10 pM. This concentration range ensures flexibility in delivering various types of cargo while maintaining control over cellular exposure and uptake efficiency. The lower limit of 1 pM allows for highly diluted cargo solutions, which may be necessary for applications requiring minimal exposure or when working with highly potent or sensitive molecules. This is particularly useful for certain signaling molecules, transcription factors, or low-abundance biomolecules where even minute concentrations can have significant biological effects, or for single molecule imaging applications requiring highly diluted label concentrations.

[0103] The upper limit of IM enables the use of highly concentrated cargo solutions, which may be necessary when working with macromolecules, nanoparticles, orformulations requiring high local concentrations for effective delivery. High concentrations are beneficial for cases where cargo uptake efficiency is inherently low, such as with certain proteins, nucleic acids, or hydrophobic compounds that require elevated levels to achieve the desired cellular response. This also ensures that sufficient cargo is available in the thin layer after spreading, optimizing interactions with the cells.

[0104] Within this range, different concentration levels can be selected based on the cargo type, cell type, and intended application. For example, nucleic acids such as plasmid DNA, siRNA, or mRNA may require concentrations in the range of 10 nM to 10 pM to achieve effective transfection without cytotoxicity. Protein-based cargos, including enzymes, growth factors, or antibodies, may require concentrations between 10 nM to 10 pM , depending on their stability and interaction with the cellular membrane. Nanoparticles and other complex formulations, such as liposomes or viral vectors, may require concentrations approaching the upper end of the range to ensure sufficient delivery. It is obvious for the skilled person that the aforementioned subranges are merely indications.

[0105] The ability to control the cargo concentration within this range provides flexibility for optimizing transfection efficiency while minimizing waste of valuable reagents. This ensures reproducibility across different applications and allows for adaptation to various research and industrial needs, such as gene editing, drug delivery, or high-throughput screening.

[0106] In a second aspect, the current invention relates to an assembly for delivering cargo to a cell. The assembly comprises a cell culturing device and an insert, wherein the cell culturing device comprises one or more compartments configured for containing one or more cells and a solution such as an aqueous solution. The solution comprises a cargo to be delivered into or onto the cells. The insert is configured to fit within at least one of the compartments.

[0107] The cell culturing device is structured to include one or more compartments, each of which is configured to hold cells adhered to or resting on a substrate. These compartments are designed to accommodate the solution containing the cargo, ensuring that the liquid remains in direct contact with the cells. The design of the compartments may vary based on application needs, ranging from standard multiwell plates and Petri dishes to specialized culture chambers that allow for precise control over the microenvironment. The compartments can be made frombiologically compatible materials such as polystyrene, glass, or biocompatible polymers, ensuring that the cells remain viable throughout the process.

[0108] In an embodiment, the cell culturing device can be a 6-well plate, 12-well plate, 24-well plate, 48-well plate, 96-well plate, 384-well plate, 1536 plate, microtube such as an Eppendorf tube, cuvette, PCR tube, cell culture flask, T-25 flask, T-75 flask, T-175 flask, Petri dish, chamber slide, microfluidic chip, cell culture insert such as Transwell insert, hanging drop plate, concave-bottom plate, ultra-low attachment plate, multi-chamber culture dish, droplet-based microfluidic culture system, microwell array, extracellular matrix-coated plate, electrospun scaffold culture system, collagen gel culture dish, PDMS culture chamber, high-throughput screening plate, roller bottle, spinner flask, rotating wall vessel bioreactor, 3D printed scaffold, nanofiber culture platform, hydrogel-based culture dish, scaffold-free 3D culture plate, air-liquid interface culture system for cell storage, suspension culture bioreactor, perfusable organoid culture system, and / or membrane-based cell culture insert.

[0109] In an embodiment, the insert comprises a substantially flat surface as a base, which is contacted with the drop of the solution and one or more vertical handles or protrusion. Said insert is configured to fit within at least one of the compartments. The insert's dimensions and geometry may be adapted based on the shape and size of the compartment, ensuring efficient coverage by the insert of the compartment bottom surface. The insert may include features that optimize its functionality, such as a flat or textured contact surface for spreading the solution, one or more handles for easy manipulation, or materials with specific properties that influence cargo delivery.

[0110] By designing the insert to interact with the compartment in a controlled manner, this assembly ensures reproducibility in the delivery process. This controlled interaction minimizes variability in exposure levels, improving the consistency of cargo uptake across different experimental conditions.

[0111] The size and proportions of the base can vary depending on the size of the substrate and the area of cells being targeted. For instance, the base may have a diameter that allows it to effectively cover the droplet while avoiding contact with areas outside the intended substrate region. The handle(s) or protrusion(s) can also vary in height and width, ensuring stability during use and compatibility with different application techniques.In an embodiment, the base will have a surface area that corresponds to at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably 100% of a corresponding surface with cells.

[0112] In an embodiment, the diameter of the base corresponds to at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably 100% of the corresponding diameter of the surface with cells.

[0113] In further embodiments of the assembly, the insert can be removable, sterilizable, and reusable, or it can be a disposable component that is replaced after each transfection procedure. This offers flexibility in research or industrial workflows. The shape of the insert can also be tailored to produce a controlled layer thickness. For example, the insert may have protrusions or ridges designed to define a specific gap relative to the cell culture substrate. As non-limiting example, such protrusions or ridges may for instance have a height of 50 or 100 pm, thereby defining such spacing between the cell surface and the surface of the insert. This may avoid direct contact between the cells and the insert, thereby avoiding damaged cells.

[0114] In an embodiment, the assembly can be adapted for high-throughput applications by configuring multiple compartments within a single device, allowing for parallel processing of multiple samples. This scalability is particularly advantageous for research, drug screening, and therapeutic applications, where reproducibility and efficiency are critical.

[0115] In an embodiment, the insert comprises a substantially flat surface configured for aligning in a substantially parallel manner with a bottom surface of the compartments. This design ensures that when the insert is positioned within the compartment, it maintains uniform contact with the solution, allowing for controlled spreading and even distribution of the cargo across the cells. By maintaining a parallel alignment, the insert minimizes variations in fluid thickness, ensuring that all cells within the compartment receive consistent exposure to the cargo.

[0116] The substantially flat surface of the insert can be manufactured with high precision to ensure minimal deviation from parallel alignment, even when slight pressure is applied during the spreading process. This reduces the risk of uneven solution distribution, which can otherwise lead to variability in transfection efficiency orinconsistent cellular responses. The insert may also be designed to self-align within the compartment, using features such as guide rails, notches, or a precisely fitted geometry that ensures proper positioning when inserted.

[0117] The choice of materials for the insert's flat surface can be tailored to optimize performance based on the type of cargo being delivered. For example, non-adhesive materials, such as polytetrafluoroethylene (PTFE) or hydrophobic-coated polymers, can be used to prevent the solution from sticking to the insert, ensuring that all of the cargo is transferred to the cells rather than interacting with the insert. Alternatively, hydrophilic coatings may be applied to promote controlled spreading and prevent localized accumulation of the solution.

[0118] Additionally, the parallel alignment of the insert's surface with the bottom surface of the compartments ensures that the thin layer formed after spreading has a controlled and uniform thickness. This is particularly important for experiments requiring high reproducibility, as variations in fluid thickness can impact cargo uptake and overall cellular responses. The configuration also facilitates compatibility with automated processing systems, ensuring that the method can be adapted for high-throughput applications without requiring manual adjustments.

[0119] In a further embodiment, the flat surface of the insert may incorporate microstructural or nanostructural features that influence the dynamics of fluid spreading. These features can help regulate how the solution interacts with the substrate, preventing undesired fluid retention on the insert or facilitating efficient displacement of the solution as it spreads into a thin layer. The insert may also be designed with adjustable height or positioning mechanisms, allowing users to finetune the distance between the insert and the cell layer, optimizing cargo delivery for specific experimental conditions.

[0120] In an embodiment, the cell culturing device is made of a translucent or transparent material, allowing for direct visualization of the cells and the solution during the cargo delivery process. The use of a material with optical clarity enables real-time monitoring of cell behavior, cargo distribution, and solution dynamics without the need for disrupting the culture environment. This transparency is advantageous for applications requiring continuous observation, such as live-cell imaging, fluorescence microscopy, and high-throughput screening assays.The transparent or translucent nature of the material allows researchers to assess the uniformity of the thin layer formed after spreading, ensuring consistent cargo distribution. It also facilitates quality control in automated systems, where optical sensors can be used to verify proper solution spreading, cargo uptake, or cell viability. The ability to observe cellular responses directly through the material eliminates the need for additional interventions, preserving the integrity of the experiment.

[0121] In another or further embodiment, where the intracellular cargo delivery is further facilitated by photoporation, said photoporation which includes laser irradiation, the insert is made from a material that is transparent or reflective to the laser light being used. In particular, when using photoporation, it is important that the insert material either does not absorb laser light or absorbs it only minimally, to prevent unintended effects such as insert destruction or photothermal damage to the cells.

[0122] The choice of material for the cell culturing device depends on the specific experimental needs. Common materials include optically clear polystyrene, borosilicate glass, polycarbonate, polymethyl methacrylate (PMMA), cyclic olefin copolymer (COC), and quartz. These materials are compatible with a range of imaging techniques, including brightfield microscopy, phase contrast, fluorescence imaging, and confocal microscopy. Glass-based devices provide excellent optical properties and chemical resistance, making them ideal for high-resolution imaging and long-term cell culture studies. Polymer-based materials, such as polystyrene or COC, offer lightweight and cost-effective alternatives, often used in disposable culture plates and multi-well formats.

[0123] In addition to enabling visualization, the translucent or transparent material can be designed to have specific properties that enhance cell culture performance. For example, surface treatments such as plasma oxidation or extracellular matrix coatings (e.g., collagen, fibronectin, or laminin) can improve cell adhesion and growth. Anti-reflective coatings may also be applied to minimize glare and enhance imaging quality. Some materials can be engineered with specific optical characteristics, such as UV transparency, which may be beneficial for experiments involving photoactivation or photoporation.

[0124] Furthermore, transparent or translucent cell culturing devices allow for the integration of optical-based detection systems, such as spectrophotometry, Raman spectroscopy, or fluorescence-based biosensors, without requiring sampleextraction. This feature enables non-invasive, real-time monitoring of cellular activity, metabolic changes, and cargo uptake efficiency, enhancing the reproducibility and accuracy of the method.

[0125] In an embodiment, where the intracellular cargo delivery is further facilitated by photoporation, said photoporation which includes laser irradiation, a material is chosen that intentionally absorbs laser light. Alternatively, light absorbing compounds, micro- or nanoparticles can be added to the material to let it absorb light. This can induce photothermal effects which can for instance permeabilize cells on the substrate, thus enabling uptake of cargo molecules. Photothermal effects include heat generation, vapor bubble generation, photoacoustic waves, plasma generation and the generation of reactive oxidative species.

[0126] Where a transparent or translucent material for the cell culturing device is used, compatibility with a wide range of analytical techniques is ensured while maintaining a stable and controlled environment for cell culture. The combination of optical clarity, chemical compatibility, and optional surface modifications makes this approach highly versatile for applications in biomedical research, drug screening, regenerative medicine, and biopharmaceutical production.

[0127] In a third aspect, the current invention relates to the use of the assembly for applying cargo to a cell by means of the method described above. The assembly, comprising a cell culturing device and an insert, enables controlled and efficient cargo delivery by facilitating the spreading of an solution containing the cargo over adherent or resting cells. The method ensures uniform exposure, resource efficiency, and reproducibility across different experimental setups.

[0128] In an embodiment, the insert applies a controlled force to transform the droplet into a thin, evenly distributed layer. The transparency or translucency of the cell culturing device allows for real-time observation of cargo delivery to or into cells, while the precise alignment of the insert ensures consistency in delivery.

[0129] This use case is adaptable to various experimental needs, including genetic modification, drug delivery, cell labeling and high-throughput screening. The system supports different cell types, cargo formulations, and delivery techniques, making it suitable for biomedical research, therapeutic development, diagnostic measurements and industrial applications. By integrating a structured assembly witha defined method, the invention enhances precision, efficiency, and scalability in cellular cargo delivery.

[0130] A person of ordinary skill in the art will appreciate that elements of the aspect of the method as described above return in the aspect of the assembly and / or of the use of the invention. Consequently, all aspects of the present invention are related. All features and related effects and / or advantages as described in one of the aspects, as described above as well as below, can relate to any of these aspects, even if they are described in conjunction with a specific aspect.

[0131] The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention.

[0132] The present invention will be now described in more details, referring to examples that are not limitative.

[0133] EXAMPLES AND DESCRIPTION OF FIGURES

[0134] The present invention will now be further exemplified with reference to the following examples. The present invention is in no way limited to the given examples or to the embodiments presented in the figures.

[0135] Figure 1 illustrates an embodiment of the method for delivering cargo (4) to cells (5) resting or adherent on a substrate (by applying a controlled force to a droplet (3) of a solution, ensuring uniform distribution and efficient uptake of the cargo. The left side of the figure depicts the initial step, where a pipette (7) is used to provide a drop (3) of 30 pL of a solution comprising a cargo (4) onto a substrate, in this case a well of a multi-well plate (2) containing cells (5). The drop, which may for instance initially be in a substantially spherical shape, partially covers the cells.

[0136] On the right side of the figure, an insert (1) is brought into contact with the drop to perform the step of applying a force in accordance with the method. The insert presses down onto the droplet, spreading the solution into a thin, substantially planar layer, ensuring complete coverage of the cells. This transformation optimizes cargocell interactions while maintaining precise control over the thin layer's thickness.By spreading the droplet in a controlled manner, the method ensures that the cargo (4), in this embodiment nucleic acids, is effectively delivered into or onto the cells, completing the method. In this embodiment, the intracellular cargo delivery is further facilitated by photoporation, enhancing the efficiency of the process. It is, however, clear that the method as shown here is not restricted to photoporation as an intracellular delivery method.

[0137] The insert is configured to fit within the well plate (2) and includes a substantially flat surface, which aligns parallel to the bottom surface of the compartment when applied onto the drop. This ensures reproducibility in cargo spreading in a thin layer, and maintains uniform exposure across all cells. In this specific embodiment, the insert includes a vertical handle (8) that facilitates handling and precise positioning of the insert onto the droplet.

[0138] As shown in the final stage, the process results in transfected cells (6), where the cargo has been successfully taken up. The structured methodology, utilizing a controlled spreading technique, addresses key limitations in traditional transfection methods by reducing reagent waste without adding cell stress or hindering reproducibility.

[0139] Example 1

[0140] An experiment was conducted to evaluate the efficiency of mRNA transfection in adherent cells and the reduction in mRNA consumption and photo-responsive particles when using an embodiment of a method and assembly according to this invention. The transfection was performed using photoporation, wherein a pulsed laser (wavelength 532 nm) and photothermal nanosensitizers (0.08 pm gold nanoparticles, between 1 and 2 pg / mL) were employed as transfection method to induce the delivery of the cargo into the cells.

[0141] In this experiment, a solution containing mRNA (0.025 mg / mL) and photothermal nanosensitizers was deposited onto adherent H1299 cells cultured in 96-well plates (lOpL), 24-well plates (30 pL), and 6-well plates (80 pL). The solution was applied using conventional pipetting techniques, after which an insert was introduced into the well to spread the droplet into a uniform thin layer over the cells. The insert comprises a substantially flat and round surface as a base, where the area of the base is approximately 95% of the area of the well. Attached to the center of the base, the insert includes a vertical handle. This ensured controlled cargo exposure while minimizing reagent waste. A pulsed laser was then applied, activating thephotothermal nanosensitizers to transiently permeabilize the cell membrane, thereby facilitating intracellular mRNA delivery.

[0142] Figure 2 presents the efficiency of mRNA transfection in the adherent cells, expressed as a percentage. The results demonstrate that transfection efficiency remains equally high across different well plate formats when using the insert compared to using the standard procedure without inserts using a large volume of cargo (96 well plate, 50 pL cargo solution). Importantly, also cell viability is maintained at an equally high level, indicating that the method does not induce significant cytotoxicity despite the reduced reagent volume.

[0143] Figure 3 shows the fold reduction in mRNA consumption achieved using the described method for various well plate formats. A significant decrease in mRNA usage is observed when inserts are applied, with greater reductions in larger well formats. Compared to the standard approach, the use of inserts enables up to a 20-fold reduction in mRNA consumption in 6-well plates, demonstrating the efficiency of the spreading mechanism in ensuring uniform cargo exposure with minimal reagent waste.

[0144] These results validate the effectiveness of the described method in delivering cargo using a small droplet of solution while maintaining high transfection efficiency and cell viability. The data further highlight the method's ability to significantly reduce reagent consumption, making it a more cost-effective and scalable alternative to conventional transfection techniques.

[0145] The present invention is in no way limited to the embodiments described in the examples and / or shown in the figures. On the contrary, methods according to the present invention may be realized in many different ways without departing from the scope of the invention.

Claims

CLAIMS1. A method for applying cargo to a cell positioned on a substrate, said method comprising:(a) providing a drop of a solution such as an aqueous solution comprising a cargo onto a substrate containing one or more cells;(b) applying a force to said drop, thereby spreading said solution into a thin layer covering said cells; and(c) delivering of said cargo into or onto said cells.

2. The method according to claim 1, characterized in that, said step (b) of applying a force to said drop is performed by contacting said drop with an insert.

3. The method according to claim 2, characterized in that, step (b) of applying a force to said drop is performed by moving and / or pressing at least a part of said insert onto said drop and / or towards said cells, thereby transforming a substantially spherical shape of said drop into a substantially planar shape of said thin layer.

4. The method according to any one of preceding claims 1-3, characterized in that, said cells are adherent to or are resting on said substrate.

5. The method according to any one of preceding claims 1-4, characterized in that, said thin layer has a thickness of between 1 pm and 1 mm, preferably of between 5 pm and 500 pm, more preferably of between 50 and 300 pm.

6. The method according to any one of preceding claims 1-5, characterized in that, said step (c) of delivering said cargo is induced by applying an intracellular delivery method, wherein the intracellular delivery method is or comprises photoporation, electroporation, lipofection, sonoporation, magnetofection, optoporation, laser-assisted transfection, or combinations thereof, preferably photoporation.

7. The method according to any one of preceding claims, characterized in that, said insert comprises a substantially flat surface which surface is contacted with said drop.

8. The method according to any one of preceding claims, characterized in that, said cargo comprises a chemical, biochemical or biological substance, molecule or particle.

9. The method according to any one of preceding claims , characterized in that, said cargo is chosen from the group consisting of DNA, plasmid DNA, cDNA, RIMA, mRNA, IncRNA, siRNA, miRNA, CRISPR-Cas9 complexes, plasmids, viral vectors, proteins, peptides, enzymes, antibodies, nanobodies, lipids, small molecules, dyes, fluorescent or luminescent markers or agents,chemotherapeutics, imaging labels such as zirconium oxide and gadolinium chelates or particles, nanoparticles, liposomes, exosomes, polysaccharides, synthetic oligonucleotides, transcription factors, cytokines, growth factors, immunomodulators, aptamers, molecular inhibitors, RIMA interference molecules, ribozymes, biosensors, synthetic transcription activators, chemical probes, therapeutic drugs, vaccine components, cell-penetrating peptides, hydrogels, magnetic particles, quantum dots, gold nanoparticles, polymeric nanoparticles, lipid nanoparticles, organelle-targeting probes, fluorescent nucleotides, metabolic tracers, and epigenetic modulators, viral- like nanoparticles, extracellular vesicles, photothermal nanoparticles, organic particles or drug loaded nanoparticles, or a combination thereof.

10. The method according to any one of preceding claims 1-9, characterized in that, said solution comprises one or more transfection aids, wherein said transfection aids are selected from physical, chemical or biological agents that enhance cellular uptake of biomolecules, preferably nanoparticles, liposomes or cationic polymers, more preferably photo responsive particles.

11. The method according to claim 10, characterized in that, said transfection aid is an organic or non-organic photo responsive particle..

12. The method according to claim 10 or 11, characterized in that, said photo responsive particles are coupled to and / or embedded in a solid support or solid material.

13. An assembly for delivering cargo to a cell, said assembly comprising a cell culturing device and an insert, wherein said cell culturing device comprises one or more compartments configured for containing one or more cells and a solution comprising a cargo to be delivered into or onto said cells, and wherein said insert comprises a substantially flat surface configured for aligning in a substantially parallel manner with a bottom surface of said compartments and wherein said insert is configured to fit within at least one of said compartments.

14. The assembly according to claim 13, characterized in that, said insert comprises a base, wherein said base has a surface area that corresponds to at least 95% with a corresponding surface with cells.

15. Use of the assembly according to any one of claims 13 or 14 for applying cargo to a cell by means of the method according to any one of claims 1-12.

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