Intracellular extraction by 2d live cell flattening (IE2d)

Abstract

The present invention relates to a method for releasing macromolecules from one or more living cells comprising (a) flattening the one or more living cells between two surfaces such that the membrane of the living cells becomes permeable or becomes more permeable for the macromolecules, wherein the surfaces that face the cells are functionalized by an antifouling coating that comprises biocompatible polymeric brushes, and (b) incubating the one or more fattened cells, wherein the macromolecules are released from the one or more living cells.

Description

[0001] Intracellular Extraction by 2D Live Cell Flattening (IE2D)

[0002] The present invention relates to a method for releasing macromolecules from one or more living cells comprising (a) flattening the one or more living cells between two surfaces such that the membrane of the living cells becomes permeable or becomes more permeable for the macromolecules, wherein the surfaces that face the cells are functionalized by an antifouling coating that comprises biocompatible polymeric brushes, and (b) incubating the one or more fattened cells, wherein the macromolecules are released from the one or more living cells.

[0003] In this specification, a number of documents are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0004] Extraction of cellular contents, in particular macromolecules (in the form of cytosol) is an essential step in studies, such as proteomics with special attention on keeping the native structure of the extracted content of live cells (also referred to herein as living cells) which very crucial in these studies. The extraction can be achieved either through cell lysis that results in breaking up the cell and releasing the cytosol or providing conditions to release partial volume of the cytosol of the live cell through the plasma membrane. The later one can be achieved either by accessing the cytosol and conducting a biopsy or by disrupting the plasma membrane which would release certain biomolecules from the cell. Considering the needs of real time extraction and preserving the native structure of the biomolecules, the recent interest of extracting cytosol content is focused on techniques that are more applicable to live cells. Moreover, the live cell techniques enable to monitor the cells for essential processes such as differentiation, cellular sequence, disease progression and effectiveness of drugs [1], In conclusion, there is the need to extract different macromolecules from the living cell such that it is possible to monitor biological functions inside the cell and / or using the extracted molecules for diagnosis or therapy purposes. Prior art methods for the extraction of cytosol from live cells can be divided into single cell and multiple cell extraction techniques. On one hand, the logic of single cell cytosol extraction relies, for example, on introducing a tube-like object inside the cell, penetrating through plasma membrane, and extracting pico-litter volumes of the cytosol. The complete overview of these methods is summarised in literature [1], These methods can be subdivided into: Nanobiopsy with glass nanopipettes [2,3], fluid force microscopy with a hollow atomic force microscope tip [4], carbon nanotube endoscopy [5] and nanostraw extraction [6,7] methods. The common aspect in all these methods relies on introducing a fine object inside the cell which can collect certain volume of the cytosol.

[0005] Also, bulk methods are available in the prior art that are applicable to populations of multiple cells. For example, such methods that reply on electroporation are reported [8,9], In electroporation, an external electrical field results in cell-membrane disruption and leaking out of the cytosol into the cell media. Although electroporation is successful in resulting in a sufficient plasma membrane disruption, there is concerns about the effect of electrical field on cytosol integrity and cell phenotype, as well as having low cell viability ratios.

[0006] In summary, the state of art provides a handful number of techniques which have been proven to successfully extract the cytosol content from one or more cells, however with low cell viability ratios. Another problem with prior art methods, such as nanobiopsy and atomic force microscopy is the complex operation and tools which requires skilled people to conduct the experiment. For example, in the nanostraw method, it requires advanced microfabrication of nano needles and a microfluidic set-up for collection of the cytosol content. In addition, the above-mentioned methods are all invasive and depending on the conduction of the experiment, the cell is damaged and that results in low cell viability. For instance, the cell viability in the case of nanostraws is reported as 5%.

[0007] Another challenge being associated with the prior art methods, is the not well-known interaction of a needle or tube with the plasma membrane

[0010] , As the plasma membrane shows soft matter property and deforms upon application of external objects, whether the plasma membrane possibly wraps around the tip of the object is not well known. Due to this fact, an extra step such an electroporation was applied to cause sufficient membrane disruption at the tip of nanotubes [7],

[0008] Considering the general picture of the available prior art methods with the advantages and disadvantages they offer, there is an ongoing need for a method having one or more of the following advantageous characteristics: • Cytosol extraction both in single and multiple cell levels applicable for live cells.

[0009] • Requiring a simple methodology with a minimum of required material and equipment.

[0010] • Being a non-invasive method, having high cell viability and minimum damage to the cytosol content or influencing the cell phenotype.

[0011] • Not requiring advanced tools and machinery, high-skilled technicians and high cost material.

[0012] This need is addressed herein.

[0013] Accordingly, the present invention relates to a method for releasing macromolecules from one or more living cells comprising (a) flattening the one or more living cells between two surfaces such that the membrane of the living cells becomes permeable or becomes more permeable for the macromolecules, wherein the surfaces that face the cells are functionalized by an antifouling coating that comprises biocompatible polymeric brushes, and (b) incubating the one or more fattened cells, wherein the macromolecules are released from the one or more living cells.

[0014] The term ..macromolecule" as used herein designates a high molecular compound (preferably in excess of 1 ,000 atoms). The macromolecule is preferably an organic macromolecule (such as DNA, RNA, protein etc.). The macromolecule Preferred examples of organic macromolecules will be described herein below.

[0015] The nature and material of the surfaces that face the cells is not particularly limited as long as the surfaces can be functionalized by an antifouling coating that comprises biocompatible polymeric brushes and results in the flattening of the cell. Preferred examples of the nature and material of the surfaces will be described herein below. The surface is generally a surface of solid material with a considerable degree of flatness to achieve a minimum distance in between two surfaces. The minimum distance assures a sufficient flattening of the cell that results in an adequate membrane disruption of the flattened cell.

[0016] The one or more cells to be used in the method of the invention are not particularly limited. Any live cell is flattened when it is confined in between the two functionalized surfaces. In this connection, it is preferred that the cells have a plasma membrane but are not covered with a rigid cell wall (such as in plant cells) that might resist the flattening of the cells. The cell may be a prokaryotic cell such as an Escherichia coli cell, but is preferably a eukaryotic cell, such as a yeast cell, fungal cell, more preferably an animal cell, even more preferred a vertebrate cell and most preferred a mammalian cell such as a human cell. The cell may be single or cells part of a cell line. Living (or “live”) cells are characterized by an intact plasma membrane barrier function. In addition, living cells are preferably characterized by one or more abilities to grow, divide, respond to their environment, and carry out metabolic activities.

[0017] Flattening is a measure of the compression of a circle or sphere along a diameter to form an ellipse or an ellipsoid of revolution (spheroid), respectively. The preferred notation for flattening is f = a - b / a, wherein a is the larger dimension (e.g. semimajor axis), and b is the smaller (semiminor axis) of the circle or sphere. In this connection it is of note that cells are generally spheroids. In this connection f is in the range of 8 to 10, depending of the cell type. For instance, an adherent cell like Dictyostelium discoideum with an average diameter of 10 pm (the values for a and b), was shown to display an average “a” value around 80 pm and a negligible “b” value (around 0.5 pm). Therefore, giving an f value around 8.

[0018] The flattening of the one or more cells has the mechanical effect which causes the membrane of the one or more living cells to become permeable or becomes more permeable for the macromolecules that are inside the one or more cells. In this respect it is to be understand that some macromolecules cannot leave the cells in the normal, non-flattened state while other macromolecules to some limited extent may leave the cells in the normal, non-flattened state (for example, through pores or extracellular vesicles) and this limited permeability can be enhanced by the flattened state.

[0019] Fouling (i.e., unwanted adhesion) is a complex and undesirable process where material from the environment, such as macromolecules, microorganisms, or suspended particles, adhere reversibly or irreversibly to a surface. The term “antifouling coating” in accordance with the present invention refers to a coating that repels proteins and cell membranes and thus block their attachment to surfaces, in the present case to the two surfaces. In particular, the “biocompatible polymeric brushes” of the antifouling coating repels proteins and cell membranes and thus blocks their attachment to surfaces, in the present case preferably to the surfaces of microscopy slides. The polymeric brushes are biocompatible which means that they do not or essentially do not interfere with the “live" status of the cells.

[0020] In accordance with the invention, a “polymeric brush” is defined as a surface coating consisting of polymers tethered to a surface or, in other terms, as polymers being attached by one end to an interface at relatively high coverage that stretch away from the interface to avoid overlapping (Milner (1991), Science, 251(4996):905-914). A polymer brush coating used for adhesion is typically <100 nm in thickness resulting in extremely low material consumption, low risk of chemical leaching, and high precision in the dimensions of the joint. Polymer brushes achieve precise control of surface chemistry, enhance material compatibility, and allow the introduction of reactive functional groups to enable strong chemical bonding. The brush may be either in a solvated state, where the tethered polymer layer consists of polymer and solvent, or in a melt state, where the tethered chains completely fill up the space available. These polymer layers can be tethered to flat substrates such as silicon wafers, or highly curved substrates such as nanoparticles. Also, polymers can be tethered in high density to another single polymer chain, noting that this arrangement has been referred to as “bottlebrush”.

[0021] Means for the functionalization of surfaces with an antifouling coating are known in the art and comprise (1) physical bonding via entanglement and (2) chemical bonding (see, for example, Buhl et al. (2020), 12(7):1475). In more detail, functionalization can be obtained either via physical interactions where the polymer brushes unify with the bulk polymer material through interpenetration and entanglement, or via chemical crosslinks where reactive groups in the polymer brush react and form covalent bonds to the bulk polymer melt.

[0022] The incubation time for step b), wherein the one or more cells are incubated the flattened state between the two surfaces is generally in the range of minutes, preferably between 1 and 60 min, more preferably between 2 min and 30 min and even more preferably between 4 and 16 min and most preferably between about 5 min and about 8 min.

[0023] The term “about” as used herein designates with increasing preference and for each occurrence separately ±20%, ±10% and ±5%.

[0024] As can be taken from the appended examples the method of the invention uses the logic of the plasma membrane disruption by cell confinement between two functionalized surfaces. It was found that such cell flattening between two surfaces disrupts the membrane to an extent that allows the release of macromolecules from the inside livings cells without or essentially without impairing the “live” status of the cells. This method is also called IE2D method herein, standing for intracellular extraction by 2D live cell flattening. By the method one or more living cells and thus either a single cell or a multiple cell population can be targeted. The sufficient membrane disruption was achieved by subjecting the living cells to confinement as 2D flattening in between two surfaces with anti-fouling coating. As the result of the membrane disruption, the live cells show permeability for release of cytosol macromolecules, such a proteins, RNA and DNA. This method offers at least the following technical advantages:

[0025] • The method is simple and requires minimum material, facilities and technical skills.

[0026] • The method is applicable to living cells with having high viability ratios over 90%.

[0027] • The method is applicable to any cell type or mixtures of different cell types with exposed plasma membrane, both adherent and suspension cells.

[0028] • The flattening step takes only few minutes (5-8 mins), therefore the cells can be subjected to extraction at room temperature without affecting the viability of them.

[0029] • The cells subjected to extraction can be cultured again and be subjected to further extraction steps, offering multiple extractions from the same cell population.

[0030] In accordance with a preferred embodiment of the invention the method further comprises after step (b) (c) removing one or both of the two surfaces and further culturing the one or more living cells for a desired period of time, and / or (d) repeating steps (a) and (b), wherein steps (c) to (d) are conducted one or more times.

[0031] As discussed herein above, cell flattening between the two surfaces does not or essentially does not impair with the “live” status of the cells. It follows that after step (b) it is possible to remove one or both of the two surfaces and further culturing the one or more living cells for a desired period of time. In this step the flattened cells return to normal shape and the cell membrane loses the permeability or the enhanced permeability for the macromolecules.

[0032] The cells can then be further cultured for a desired period of time in step (c), wherein the cells can, for example, again produce the macromolecules that were released in step (b). Thereafter in step (d) steps (a) and (b) can be repeated. These steps (a) to (d) can be repeated one or more times, so that macromolecules can be released from the living cells repeatedly, in a periodic and controllable manner. This level of control is a technical advantage of the method of the invention.

[0033] In accordance with another preferred embodiment of the invention the one or more living cells comprise at least two living cells and in step (b) a part of the released macromolecules of the first cell are taken up by the second cell and vice versa.

[0034] In this connection it is to be understood that the flattening of the cells does not only result in or enhance the release of macromolecules from the cells but also allows allows the delivery of macromolecules into different cells, It follows that the method of the above preferred embodiment can also be characterized as a method for exchanging macromolecules between different cells. Such an exchange of macromolecules between different cells can advantageously be used, for example, in cell-to- cell signaling studies, cell-by-cell activation studies or intercellular communication studies.

[0035] In accordance with a further preferred embodiment of the invention in step (b) cytosol is released from the one or more living cells and the released macromolecules are within said cytosol.

[0036] As discussed herein above, the flattening of the cells results in the permeability or enhanced permeability of the cells. This permeability or enhanced permeability is non-specific, which means that the flattening of the cells generally results in the permeability or enhanced permeability of the cell membrane for macromolecules but not for any specific macromolecule.

[0037] The macromolecules are preferably and generally released from the cells as cytosol. The cytosol (also known as cytoplasmic matrix or ground plasm) is one main liquid found inside cells (intracellular fluid (ICF)). The cytosol is a complex mixture of macromolecules dissolved in water. In addition to the macromolecules the cytosol comprises ions, such as sodium and potassium ions. The ion concentration and the macromolecule composition of the cytosol are different to those in the extracellular fluid. These differences are important in many biological processes, such as osmoregulation, cell signaling, and the generation of action potentials in excitable cells such as endocrine, nerve and muscle cells.

[0038] In accordance with a yet further preferred embodiment of the invention the method further comprises separating the released macromolecules from the one or more living cells.

[0039] Means and methods for separating the released macromolecules from the one or more living cells are known in the art and comprise, for example, cell sorting (e.g. by FACS), cell filtration (in which case the pore size is such that cells are retained in the filter), or cell sedimentation by centrifugation.

[0040] In accordance with a more preferred embodiment of the invention the released macromolecules or selected released macromolecules are isolated from the cells by a membrane with nano- or micropores, wherein the membrane with nano- or micropores is preferably placed between the one or more cells and one of the two surfaces, such that released macromolecules that fit though the nano- or micropores of the membrane can pass the membrane and wherein one or more spacers keep the membrane with nano- or micropores away from the opposite one of the two surfaces so that a space is generated that can harbor the released macromolecules that passed the membrane.

[0041] The membrane with nano- or micropores has a pore size that retains the cells but allows the released macromolecules or the released macromolecules having a preselected size to pass the membrane. Thereby the macromolecules are separated from the one or more cells.

[0042] Suitable membrane materials with nano- or micropores are available in the prior art. The membrane material can be chosen based on the type of macromolecules indented for filtering.

[0043] For instance and preferably, the following microporous materials can be used:

[0044] 1. Porous glass, preferably having a pore size in the range of about 0.2 pM to about 5 pM. Microporous glass products can be obtained for example, by heat treatment and the filtration of alkaliborosilicate glass (Na2O-B2Os-SiO2). During the heat treatment, the silica-rich phase and the alkali phase take place separately, and silica-rich glass with connected pores can be prepared via a percolation of the alkali phases with hydrochloric acid. The average pore sizes are 1-10 nm, with surface areas of 50-500 m2 / g. If further percolation is conducted, the pore sizes can be adjusted within the range of 10 nm-20 pm; see P.S. Liu, G.F. Chen, in Porous Materials, 2014.

[0045] 2. Microporous polymeric materials such as PMMA (Poly(methyl methacrylate)), wherein the polymeric material has preferably a pore size of about 0.3 - about 0.7 pM.

[0046] For instance and preferably, the following nanoporous materials can be used:

[0047] 1. Nanoporous Anodic AI2O3, SiC>2 (ordered, straight), preferably having a pores size of > about 10 nm.

[0048] 2. Nanoporous polymers, such as PC (polycarbonate), PE (polyethylene), wherein the nanoporous polymers preferably have a pore size of > about 20 nm.)

[0049] A summary of the suitable nanoporous membrane materials can be found, for example in the review Adiga et al. (2009), “Nanoporous membranes for medical and biological applications”, https: / / d0i.0rg / l 0.1002 / wnan.50)

[0050] It is to be understood that the membrane will generally be only between the cells and of one of the two surfaces, either the top or bottom surface. Both options can be used. As spacers, for example, pillars or posts can be used that are connected to the membrane and / o the opposite surface.

[0051] In accordance with an even more preferred embodiment of the invention, a microfluidic flow is applied to the space between the membrane with nano- or micropores and the surface opposite said membrane, so that that macromolecules that passed the membrane can be collected.

[0052] By the microfluidic flow the released macromolecules that passed the membrane can be transported outside of the areas between the membrane and the surface that faces the membrane. The macromolecules may be collected, for example, in a vial.

[0053] In accordance with another preferred embodiment of the invention, the method further comprises formulating the released macromolecules into a composition, preferably a diagnostic or pharmaceutical composition.

[0054] The composition comprises the released macromolecules and at least one further compound, such as a diluent, excipient, carrier or adjuvant.

[0055] A diagnostic composition is a composition which is for the identification of a disease or condition in a subject and a pharmaceutical composition is a composition which is for the prevention or treatment of a disease or condition in a subject.

[0056] Examples of suitable carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions, organic solvents including DMSO etc. Compositions comprising such carriers can be formulated by well-known conventional methods.

[0057] These pharmaceutical or diagnostic compositions can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The therapeutically effective amount for a pharmaceutical composition or the diagnostically effective amount of a diagnostic composition for a given situation will readily be determined by routine experimentation and is within the skills and judgement of the ordinary clinician or physician. In accordance with a preferred embodiment of the invention, the method further comprises isolating the released macromolecules or selected released macromolecules.

[0058] Also means and methods for isolating the released macromolecules or selected released macromolecules are known in the art. For instance, for isolating the released macromolecules also filtration can be used (in which case the pore size is such that macromolecules are retained in the filter, for example in the form a gel filter). Selected released macromolecules may be isolated, for example, by nucleic acid (e.g. DNA and / or mRNA) or protein extraction kits or nucleic acid (e.g. DNA and / or mRNA) or protein gels. Selected nucleic acids or proteins may be isolated, for example, by specific nucleic acid hybridization probes or antibodies.

[0059] In accordance with another preferred embodiment of the invention, the one or more living cells comprise different cell types.

[0060] Since different cell types comprise different compositions of macromolecules the use of different cell types in the method of the invention can also be used to generate compositions of released macromolecules with desired properties. For instance, one cell type may release an extracellular receptor and the other cell type may release the soluble receptor or one cell type may release one part of a FRET pair and the other cell type may release the other part of a FRET pair.

[0061] In accordance with a further preferred embodiment of the invention, the one or more living cells comprise one or more adherent cells and / or one or more suspension cells.

[0062] The method of the invention can be used to flatten both, adherent cells and suspension cells.

[0063] The method is particularly beneficial for suspension cells, because they generally tend to not adhere to surfaces as contrasted to adherent cells. Therefore, they tend to cluster inside the cell medium as floating spheroids that is not convenient in the case of methods introduced in the art. The IE2D method is capable of flattening of cell spheroids as a cluster of multiple cells. Flattening does not require or result in adhering of the cells to the surfaces, because the anti-fouling coating avoids the adhesion.

[0064] In accordance with a preferred embodiment of the invention, the two surfaces are made of glass, quartz, silicon wafers, polyolefin (preferably cyclic olefin copolymer (COC) or cycloolefin polymers (COP)), plasma-activated polymers, and / or metal oxides (preferably Ta2Os, indium tin oxide (ITO), TiO2, ZrO2).

[0065] The above materials for the two surfaces are preferred because these materials have a considerably flat surface with minimum roughness and can be functionalized by an antifouling coating that comprises biocompatible polymeric brushes.

[0066] In accordance with another preferred embodiment of the invention, the two surfaces are microscopy slides, preferably glass microscopy slides.

[0067] The microscopy slides are used in the examples. Microscopy slides at various sizes and of various thickness are commercially available. Microscopy slides are usually made of optical quality glass, such as soda lime glass or borosilicate glass, but specialty plastics are also used.

[0068] In accordance with a further preferred embodiment of the invention, the two surfaces have a size of 24 mm ± 50% x 50 mm ± 50%, a thickness of 0.170 mm ± 50%, and / or a weight of 0.605 ± 50% gram.

[0069] Surfaces in the form of microscopy slides with the above size and thickness are illustrated by the examples. Together with the brush-like structures they were found to be particularly suitable to flatten various eukaryotic cell types, in particular neural stem cells, Dictyostelium discoideum cells, fibroblasts, HeLa cells, cardiomyocytes and epithelial cells. They impose a pressure on the cells that flattens the cells without destroying the cells and allowing the release of macromolecules from the cells.

[0070] In accordance with another preferred embodiment of the invention, the cells are within a medium and preferably the medium comprises objects, preferably beads, wherein the objects allow for determining the degree of flattening of the cells by keeping the two microscopy slides apart from each other at a desired distance.

[0071] As discussed above, the method of the invention is applied to living cells and the medium is helpful to keep the cells alive. Accordingly, the medium is preferably a cell maintenance medium and preferably a growth medium. In a maintenance medium the cells stay alive and in a growth medium the cells can survive and divide.

[0072] As also discussed above, the microscopy slides (e.g. their size and thickness) can be selected such that they impose a pressure on the cells that flattens the cells without destroying them. As an alternative the degree of flattening can be set by objects, preferably beads. The degree of flattening in turn also adjusts the permeability of the cell membrane for the macromolecules, noting that a high degree of flattening results in a high degree of permeability.

[0073] The objects, preferably beads are micro-objects, microbeads having a height or diameter being less than the height or diameter of the cells to be flattened. The micro-objects or microbeads can be of any suitable material such a polymeric material (silicone) or a metal / metal oxide (e.g. gold or iron oxide).

[0074] In accordance with a preferred embodiment of the invention, the biocompatible polymeric brushes of the antifouling coating are (a) linear brushes comprising polyethylene of glycol (PEG), lubricin, polyoxazolines, polyglycerol, dendrons, polyvinylpyrrolidone (PVP), polysaccharides, polypeptoids, polyacrylamide and / or zwitterionic polymers; and / or (b) bottlebrushes comprising polyvinylpyrrolidone (PVP), poly[(oligoethylene glycol) methyl ether methacrylate] (POEGMA), member of the polyoxazoline family, poly(2-methyl-2- oxazoline) (PMOXA), and / or thiol end-capped poly(methacrylic acid)-g-poly(2-methyl-2- oxazoline) (PMAA-g-PMOXA).

[0075] The above preferred biocompatible polymeric brushes are reviewed, for example, in Maan et al. (2020), Advanced functional materials, 30(32), 2000936.

[0076] There are many alternative coatings and any one of the above preferred antifouling coatings that are all biocompatible can be used. Another aspect to be considered is the hydrophilicity of the coating that is necessary for spreading of the cell solution in between two glasses. The flattening happens due to high hydrophilicity of the coated surfaces that result in strong capillary flow. Also, in this respect the above biocompatible polymeric brushes are expected to be particularly useful.

[0077] In accordance with another preferred embodiment of the invention the antifouling coating that comprises biocompatible polymeric brushes is a co-polymer having a poly(L-lysine) backbone and poly (ethylene glycol) brushes.

[0078] This co-polymer is also known as PLL-g-PEG. PLL-g-PEG is a random graft co-polymer with a poly(L-lysine) backbone and poly(ethylene glycol) side-chains. The PLL backbone interacts electrostatically with the substrate, while the sidechains extend from the surface to form a densely packed polymeric brush. A backbone polymer is generally used when the main polymer does not covalently bind to the surface. For instance, in PLL-g-PEG, as the glass surface is negatively charged the poly- (L-Lysine) is used which has positive surface charge. This results in covalent binding. In all the bottlebrushes wherever the name indicates -g-, it refers to grafted polymers. The first polymer is the backbone and the second one is the brush. For example, in the case of PMMA-g-PMOXA, PMMA is the backbone polymer.

[0079] PLL-g-PEG can adhere to surfaces, such as microscopy slides by electrostatic adsorption from aqueous buffers. The PLL-g-PEG coating has a brush-like structure well characterized by protein absorption and friction coefficient 9 measurements.

[0080] PLL-g-PEG is preferably applied as a coating solution. The coating solution preferably has a final PLL-g-PEG concentration 0.1 mg / ml ±50% in 10 mM ±50% HEPES (pH 7.4, at room temperature). The coating solution is preferably applied as a drop on a parafilm layer and the coating surfaces are put on the drop. Therefore, the drop in between the parafilm layer and the coating surface expands and covers whole surface which is normally incubated for 30 min ±50%, preferably in a ventilated hood. Remaining liquid may be removed and slides can be dried by air flow.

[0081] In a more preferred embodiment, the poly(L-lysine) forming the backbone has a molecular weight of 20kDa ± 50% and / or the poly (ethylene glycol) brushes have a molecular weight in the range of 2kDa ± 50% and 5kDa ± 50%.

[0082] The poly(L-lysine) having a molecular weight of 20kDa ± 50% has with increasing preference a molecular weight of 20kDa ± 40%, 20kDa ± 30%, 20kDa ± 20%, 20kDa ± 10%, and 20kDa ± 5%.

[0083] Similarly, the poly (ethylene glycol) brushes having a molecular weight in the range of 2kDa ± 50% and 5kDa ± 50% have with increasing preference a molecular weight in the range of 2kDa ± 40% and 5kDa ± 40%, 2kDa ± 30% and 5kDa ± 30%, 2kDa ± 20% and 5kDa ± 20%, 2kDa ± 10% and 5kDa ± 10%, and 2kDa ± 5% and 5kDa ± 5%. In another more preferred embodiment, the copolymer comprises or has the formula:

[0084] In the above formula m refers to the PEG molecular weight, 5 kDa j refers to the grafting ratio, ~3. and k refers to the PLL molecular weight, 20 kDa.

[0085] In a preferred embodiment, the method comprises prior to (a):

[0086] (1) activating the two surfaces in oxygen plasma, preferably for 3 min ± 50% at 0.5 mbar ± 50%;

[0087] (2) coating the activated two surfaces with the antifouling agent, preferably the co-polymer of poly(L-lysine) and poly (ethylene glycol), wherein the co-polymer preferably is used at a concentration 0.1 mg / ml± 50% in a buffer, wherein the buffer is preferably 10 mM ± 50% HEPES; and

[0088] (3) optionally drying the coated two surfaces, preferably in a ventilated hood.

[0089] Surface contaminations, in particular the residues remaining after mechanical and wetchemical cleaning, are often organic. In many cases, these are residues of oils, greases, release agents, and silicones which many solvents cannot remove completely. If these substances remain on the surface, they impair all downstream processing steps and have a particularly negative effect on all types of bonding and coating. These substances can be removed residue-free in an oxygen plasma, often also an air plasma. Excited oxygen molecules and radicals are highly reactive and form very stable bonds.

[0090] The UV radiation of the radiation splits the polymer chains of the organic residues. Oxygen radicals immediately occupy the released bonds and prevent the polymer fragments from reuniting. In this way, short-chain volatile substances which can be sucked off by the vacuum pump are increasingly formed from the macromolecules.

[0091] In the above preferred embodiment the ± 50% are with increasing preference ± 40%, ± 30%, ± 20%, ± 10% and ± 5%.

[0092] In a more preferred embodiment, the method further comprises prior to (1) the sterilization of the two surfaces, preferably by UV light.

[0093] UV light can optionally be combined with ethanol and preferably only UV light is used.

[0094] Alternatively, or in addition the surfaces, such as microscopy slides can be sterilized or cleaned by one or more and preferably all of

[0095] (i) washing in 100% ethanol (e.g. for 10min ±50%) and rinsing in deionized water.

[0096] (ii) sonication in acetone (e.g. for 30 min ±50%) optionally followed by incubated in 96% ethanol (e.g. for 10 min ±50%), and

[0097] (iii) incubation in a Hellmanex III solution (Hellma Analytics) (e.g. 2% ±50% for 2h ±50%).

[0098] The Hellmanex is generally a step which is also beneficial for better imaging through the surfaces, such as microscopy slides. In this connection it is to be understood that although the Hellmanex step improves the optical properties of the glass cover slips it is mostly used in the appended examples as a cleaning agent. While the optical improvement is beneficial, the main purpose of the optional Hellmanex step is cleaning. According to the company description of the product, “Hellmanex® III, significantly reduces the surface tension of water. The Hellmanex aqueous solution helps with the removal of dirt particles, and its high emulsifying and dispersing capabilities prevent the redeposition of the loosened particles. Special surface-active substances facilitate the residue-free rinsing of the optical components once they have been cleaned, (see https: / / www.hellma.com / en / laboratory- supplies / cuvettes / hellmanex-cleaning-concentrate / )” In the above preferred embodiment the ± 50% are with increasing preference ± 40%, ± 30%, ± 20%, ± 10% and ± 5%.

[0099] In a preferred embodiment, the macromolecules are proteins, peptides, nucleic acids molecules, carbohydrates, lipids, nanogels, macrocycles or a combination thereof.

[0100] The term “protein” as used herein interchangeably with the term “polypeptide” describes linear molecular chains of amino acids, including single chain proteins or their fragments, containing at least 50 amino acids. The term “peptide” as used herein describes a group of molecules consisting of up to 49 amino acids, whereas the term “polypeptide” (also referred to as "protein") as used herein describes a group of molecules consisting of at least 50 amino acids. The term “peptide” as used herein describes a group of molecules consisting with increased preference of at least 15 amino acids, at least 20 amino acids at least 25 amino acids, and at least 40 amino acids. The group of peptides and polypeptides are referred to together by using the term "(poly)peptide". (Poly)peptides may further form oligomers consisting of at least two identical or different molecules. The corresponding higher order structures of such multimers are, correspondingly, termed homo- or heterodimers, homo- or heterotrimers etc. Furthermore, peptidomimetics of such proteins / (poly)peptides where amino acid(s) and / or peptide bond(s) have been replaced by functional analogues are also encompassed by the invention. Such functional analogues include all known amino acids other than the 20 gene-encoded amino acids, such as selenocysteine. The terms “(poly)peptide” and “protein” also refer to naturally modified (poly)peptides and proteins where the modification is effected e.g. by glycosylation, acetylation, phosphorylation and similar modifications which are well known in the art.

[0101] The term “nucleic acid molecule” in accordance with the present invention includes DNA, such as cDNA or double or single stranded genomic DNA and RNA. In this regard, "DNA" (deoxyribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and thymine (T), called nucleotide bases, that are linked together on a deoxyribose sugar backbone. DNA can have one strand of nucleotide bases, or two complimentary strands which may form a double helix structure. "RNA" (ribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and uracil (II), called nucleotide bases, that are linked together on a ribose sugar backbone. RNA typically has one strand of nucleotide bases, such as mRNA. Included are also single- and double-stranded hybrids molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA. The nucleic acid molecule may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Nucleic acid molecules, in the following also referred as polynucleotides, may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Further included are nucleic acid mimicking molecules known in the art such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers. Such nucleic acid mimicking molecules or nucleic acid derivatives according to the invention include phosphorothioate nucleic acid, phosphoramidate nucleic acid, 2’-O-methoxyethyl ribonucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA), peptide nucleic acid (PNA) and locked nucleic acid (LNA) (see Braasch and Corey, Chem Biol 2001, 8: 1). LNA is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2’-oxygen and the 4’-carbon. Also included are nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil. A nucleic acid molecule typically carries genetic information, including the information used by cellular machinery to make proteins and / or polypeptides. The nucleic acid molecule of the invention may additionally comprise promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like.

[0102] A carbohydrate refers to any of the group of organic compounds consisting of carbon, hydrogen, and oxygen, usually in the ratio of 1 :2:1 , hence the general formula: Cn(H2O)n. Carbohydrates are the most abundant among the major classes of biomolecules.

[0103] Lipids are a broad group of organic compounds which include fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. Lipids be in the form of exosomes. Exosomes are lipid packages which can carry a variety of different molecules including RNA (Alexander et al. (2015), Nat Commun; 6:7321).

[0104] Nanogels are three-dimensional hydrogel materials in the nanoscale size range formed by crosslinked swellable polymer networks with a high capacity to hold water, without actually dissolving into the aqueous medium. Nanogels can be composed of a variety of naturally occurring polymers, synthetic polymers or a combination thereof. Their characteristics such as size, charge, porosity, amphiphilicity, softness, and degradability can be fine-tuned by varying the chemical composition of the nanogels. They are mostly spherical particles but the current advancement in synthetic strategies allow for the fabrication of nanogels of different shapes. They can be also designed to have either a core-shell or a core-shell-corona structure, with at least one of the layers crosslinked for structural integrity. Being mostly hydrophilic in nature, nanogels are highly biocompatible with a high loading capacity for guest molecules and their unique physical properties offer them distinct advantages over other types of nanomaterials for biomedical applications.

[0105] Macrocycles are molecules or ions containing a ring of seven or more, preferably twelve or more atoms. Classical examples include the crown ethers, calixarenes, porphyrins, and cyclodextrins.

[0106] In a preferred embodiment, the macromolecules are particles.

[0107] The particles are generally microparticles. Microparticles are particles having a site of 100 m or below. Commercially available microparticles are available in a wide variety of materials, including ceramics, glass, polymers, and metals. Microparticles encountered in daily life include pollen, sand, dust, flour, and powdered sugar. The microparticles are preferably microspheres or microbeads. The surface of the particles can be functionalized with the above-described macromolecules. Also virus or virus-like particles are encompassed by the term “particles”.

[0108] As regards the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

[0109] Similarly, and also in those cases where independent and / or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1, a dependent claim 2 referring back to claim 1 , and a dependent claim 3 referring back to both claims 2 and 1 , it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1. In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1 , of claims 4, 2 and 1, of claims 4, 3 and 1 , as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

[0110] The figures show.

[0111] Figure 1 : Result of silver staining of proteins in polyacrylamide gel comparing flattened cells and the surrounding buffer with non-flattened cells and their surrounding buffer. Numbers 1 to 5 correspond to standard, non-confined cells, confined cells, buffer of the non-confined cells and buffer of the confined cells, respectively.

[0112] Figure 2: Schematics of the experiments of the cell-cell communication, a) shows the mechanism of the proteins release from the flattened cell confined in between two PII-g-PEG functionalized glasses, b) Simultaneous release of delivery of the proteins from one cell type to another cell type, the difference of the cell types is shown as green and red colors.

[0113] Figure 3: Schematics of the setup for real-time and selective extraction of macromolecules such as proteins and RNA from the flattened cells, a) shows a 3D schematic highlighting the use of solid porous membrane under the cells, b) shows the logic of real-time protein or RNA extraction and selection through the porous membrane, a slow and continuous flow under the membrane would result in efficient extraction and collection of the macromolecules, c) shows the gel concept for sorting of the real-time sorting of extracted macromolecules from the cells. The yellow color layer represents a gel with sorting pore sizes to trap different proteins in different depth of the gel.

[0114] The examples illustrate the invention.

[0115] Example 1 - Detection of proteins extracted from the cells

[0116] In order to confirm that the live cell flattening results in release of proteins from the cells, silver staining of proteins in polyacrylamide gel method was conducted to detect the proteins concentration in the cell buffer around the flattened cells. The cells (AX2 wild type) were confined in between the glass slides with PLL-g-PEG coating for 8 mins. The cells were collected in a tube and were subjected to centrifugation. The medium above the cells was collected and subjected to the measurement. The result is shown in Figure 1. Numbers 1 to 5 correspond to standard, non-confined cells, confined cells, buffer of the non-confined cells and buffer of the confined cells, respectively. The lines 2-3 are considered as the control experiment to compare the extracted proteins from lysed cells. Comparing of the lines 4 and 5, shows presence of more protein density in confined buffer (the buffer around the cell which were flattened), as well as distinct proteins that do not exist in the non-confined cell buffer. Therefore, the flattening is capable of extraction proteins that normally the live cells would not release them in standard cell culture conditions.

[0117] Example 2 - Cell-Cell Communication; simultaneous intracellular extraction and delivery

[0118] The membrane-disruption results in two-way intracellular transport, extraction and delivery. In other words, a cell with disrupted membrane at the same time that releases molecules would take in external molecules inside the cell. The intracellular delivery of external macromolecules inside the live cells was discussed in ID2D invention. Therefore, combining these two findings was considered to prove the release of certain proteins from the live cell by confirming the intracellular delivery of them to other cells. As a model cell, Dictyostelium discoideum (D.d) amoebae cells either in the wild-type or genetically modified form depending on the experiment were used. Figure 2 shows the schematics of the experiments which can be described as:

[0119] • One-way cell-cell communication: collection of extracted proteins from a flattened cell and introduction of them to another cell population

[0120] • Bidirectional cell-cell communication: simultaneous releasing and up-taking of the proteins in adjacent flattened cells

[0121] Example 2.1 - One-way cell-cell communication

[0122] In order to confirm the release of proteins from the cells subjected to 2D confinement, the D.d cells that were previously transmitted by the Myosin-GFP vector were used. The cells were confined as 2D and the cell buffer was collected after cell confinement for 8 mins. Afterwards, the collected buffer was mixed with wild type D.d cells and were confined to flattening to conduct the delivery, as reported in ID2D method.

[0123] Two different methods were used to confirm the presence of proteins in the wild type D.d cells, namely: (i) using selection markers which are antibodies that result in selection of the having the delivered proteins, and (ii) the fluorescent microscopy methods were used to capture the florescent signals. The results of selection marker experiment and microscopy observations confirmed delivery of the proteins inside the wild-type cells. Therefore, it confirms the release of proteins from the first cell line (genetically modified with Myosin-GFP vector).

[0124] Example 2.2 - Bidirectional cell-cell communication

[0125] Two different cell lines which were genetically modified to express fluorescent proteins were used to study the spontaneous release and delivery of the proteins in between them. For this purpose, AX2-Actin-GFP and AX2- Lim-mRFP cell lines were mixed together and confined for 8 mins. The cell was collected and were subjected to the both selection markers of Actin- GFP and Lim-RFP. The cell attachment and proliferation confirmed the bidirectional cell-cell communication and also the cells were double checked with fluorescent microscope to visualize the presence of fluorescent molecules.

[0126] Example 3 - Detection of extracted RNA

[0127] The live cells release normally different types of RNA and DNA molecules. As the 2D flattening has shown release of proteins, it suggested the release of RNA and DNA molecules from the live cells. Preliminary experiments were conducted to analytically detect the concentration differences of RNA and DNA in the buffer collected from confined and nonconfined cells. Spectrophotometry method was used for these measurements. The result showed higher concentration of RNA and DNA in the buffer collected from the confined cells. Nevertheless, the ongoing experiments such as RNA sequencing are under progress to specify the extract type of the RNA molecules.

[0128] Example 4: Preparation of cover glass slides

[0129] Microscopy slides (Zeiss company, 24*50 mm, thickness of 0.170 mm ± 0.005 mm and weight of 0.605 ± 0.001 gr) were cleaned by washing with 100% ethanol for 10min and rinsing in deionized water. They were further sonicated in acetone for 30 min and followed by incubation in 96% ethanol for 10 min. The last step was 2h incubation in a 2% Hellmanex III solution (Hellma Analytics). Finally, slides were extensively washed in deionized water and dried with a filtered airflow. All cleaning procedures were performed at room temperature. The cleaned slides were immediately activated in oxygen plasma (FEMTO, Diener Electronics, Germany) for 3 min at 0.5 mbar. The coating solution with a final concentration 0.1 mg / ml of the Poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) (SuSoS AG, Switzerland) in 10 mM HEPES (pH 7.4, at room temperature) was prepared. The coating solution was applied as drops on a clean surface and the activated glass slides were put on it and incubated for 30 min in a ventilated hood. Remaining liquid was removed and slides were dried by air flow.

[0130] Example 5 - Future Experiments

[0131] Example 5.1 - RNA sequencing and mRNA detection

[0132] It is planned to acquire a full RNA sequencing of the RNA molecules extracted from the cell. This will enable to distinguish the different types of RNA, such as mRNA, non-coding RNA, micro RNA and etc. mRNA is a new avenue in medical researches for potential therapeutical applications which is so far was used in Covid vaccines

[0011] ,

[0133] Example 5.2 - Full detection and classification of the extracted proteins by Mass spectrometry and western blot

[0134] It is planned to conduct mass spectrometry of extracted proteins from different cell types. These measurements will provide exact type of proteins and their quantity in the extracted buffer from flattened cells. It is known that the cells under shock, for example heat shock, produce higher amount of proteins and applying a shock method is normally used to produce proteins from live cells. It is hypothesised that mechanical stress from 2D flattening could result in higher production of certain proteins inside the cell which is not studied in literature.

[0135] Example 5.3 - Real-time extraction and collection by a microfluidics device

[0136] Figure 3, a,b shows the schematics of a suitable device for real time collection of proteins and RNA / DNA macromolecules from the cells. The design uses a third substrate which nano or micro sized pores. The pores would be used for collection of the extracted macromolecules from the cells in a real-time manner, a continuous and slow flow under the membrane would enable collection of the macromolecules. In this setup the pore size can be adjusted to permit only specific proteins sizes and therefore having a selection mechanism of the extracted proteins (Figure 3.b). The design could include the mechanical system (such as including beads to control the flattening level) to adjust the flattening degree and study the relation between the protein extraction as a function of the cell flattening level. The implementation of a microfluidic flow will offer a continuous sorting possibility that is shown schematically in Figure 3.c. Certain gels are commercially used with the help electrophoresis to sort proteins or RNA molecules based of their molecular weight and size. It is foreseen to engineer a setup allowing real-time electroporation like sorting of the extracted macromolecules from live flattened cells.

[0137] References

[0138] [1] Stuart G Higgins and Molly M Stevens. Extracting the contents of living cells. Science, 356(6336):379-380, 2017.

[0139] [2] Paolo Actis, Michelle M Maalouf, Hyunsung John Kim, Akshar Lohith, Boaz Vilozny, R Adam Seger, and Nader Pourmand. Compartmental genomics in living cells revealed by single-cell nanobiopsy. ACS nano, 8(1):546-553, 2014.

[0140] [3] Binoy Paulose Nadappuram, Paolo Cadinu, Avijit Barik, Alexander J Ainscough, Michael J Devine, Minkyung Kang, Jorge Gonzalez-Garcia, Josef T Kittier, Keith R Willison, Ramon Vilar, et al. Nanoscale tweezers for single-cell biopsies. Nature nanotechnology, 14(1):80- 88, 2019.

[0141] [4] Orane Guillaume-Gentil, Rashel V Grindberg, Romain Kooger, Livie Dorwling- Carter, Vincent Martinez, Dario Ossola, Martin Pilhofer, Tomaso Zambelli, and Julia A Vorholt. Tunable single-cell extraction for molecular analyses. Cell, 166(2): 506-516, 2016.

[0142] [5] Riju Singhal, Zulfiya Orynbayeva, Ramalingam Venkat Kalyana Sundaram, Jun Jie Niu, Sayan Bhattacharyya, Elina A Vitol, Michael G Schrlau, Elisabeth S Papazoglou, Gary Friedman, and Yury Gogotsi. Multifunctional carbon-nanotube cellular endoscopes. Nature nanotechnology, 6(1):57-64, 2011.

[0143] [6] Xi Xie, Alexander M Xu, Sergio Leal-Ortiz, Yuhong Cao, Craig C Garner, and Nicholas A Melosh. Nanostraw-electroporation system for highly efficient intracellular delivery and transfection. ACS nano, 7(5):4351-4358, 2013.

[0144] [7] Yuhong Cao, Martin Hjort, Haodong Chen, Fikri Birey, Sergio A Leal-Ortiz, Crystal M Han, Juan G Santiago, Sergiu P Pa. sea, Joseph C Wu, and Nicholas A Melosh. Nondestructive nanostraw intracellular sampling for longitudinal cell monitoring. Proceedings of the National Academy of Sciences, 114(10):E1866-E1874, 2017.

[0145] [8] Yihong Zhan, Chen Sun, Zhenning Cao, Ning Bao, Jianhua Xing, and Chang Lu. Release of intracellular proteins by electroporation with preserved cell viability. Analytical chemistry, 84(19):8102-8105, 2012.

[0146] [9] Gen He, Chengduan Yang, Tian Hang, Di Liu, Hui-Jiuan Chen, Ai-hua Zhang, Dian Lin, Jiangming Wu, Bo-ru Yang, and Xi Xie. Hollow nanoneedle-electroporation system to extract intracellular protein repetitively and nondestructively. ACS sensors, 3(9): 1675-1682, 2018.

[0010] Giro Chiappini, Enrica De Rosa, JO Martinez, Xuewu Liu, Joe Steele, MM Stevens, and Ennio Tasciotti. Biodegradable silicon nanoneedles delivering nucleic acids intracellularly induce localized in vivo neovascularization. Nature materials, 14(5): 532-539, 2015.

[0011] Ann J Barbier, Allen Yujie Jiang, Peng Zhang, Richard Wooster, and Daniel G Anderson. The clinical progress of mrna vaccines and immunotherapies. Nature biotechnology, 40(6): 840-854, 2022.

Claims

CLAIMS1. A method for releasing macromolecules from one or more living cells comprising(a) flattening the one or more living cells between two surfaces such that the membrane of the living cells becomes permeable or becomes more permeable for the macromolecules, wherein the surfaces that face the cells are functionalized by an antifouling coating that comprises biocompatible polymeric brushes, and(b) incubating the one or more fattened cells, wherein the macromolecules are released from the one or more living cells.

2. The method of claim 1 , wherein the method further comprises after step (b)(c) removing one or both of the two surfaces and further culturing the one or more living cells for a desired period of time, and / or(d) repeating steps (a) and (b), wherein steps (a) to (d) are conducted one or more times.

3. The method of claim 1 or 2, wherein the one or more living cells comprise at least two living cells and wherein in step (b) a part of the released macromolecules of the first cell are taken up by the second cell and vice versa.

4. The method of any one of claims 1 to 3, wherein in step (b) cytosol is released from the one or more living cells and wherein the released macromolecules are within said cytosol.

5. The method of any one of claims 1 to 4, further comprising separating the released macromolecules from the one or more living cells.

6. The method of claim 5, wherein the released macromolecules or selected released macromolecules are separated from the one or more cells by a membrane with nano- or micropores, wherein the membrane with nano- or micropores is preferably placed between the one or more cells and one of the two surfaces, such that released macromolecules that fit though the nano- or micropores of the membrane can pass the membrane, and wherein one or more spacers keep the membrane with nano- or micropores away from the opposite one of the two surfaces so that a space is generated that can harbor the released macromolecules that passed the membrane.

7. The method of claim 6, wherein a microfluidic flow is applied to the space between the membrane with nano- or micropores and the surface opposite said membrane, so that that macromolecules that passed the membrane can be collected.

8. The method of any one of claims 1 to 7, further comprising isolating the released macromolecules or selected released macromolecules.

9. The method of any one of claims 1 to 8, further comprising formulation the released macromolecules into a composition, preferably a diagnostic or pharmaceutical composition.

10. The method of any one of claims 1 to 9, wherein the one or more living cells comprise different cell types.

11. The method of any one of claims 1 to 10, wherein the one or more living cells comprise one or more adherent cells and / or one or more suspension cells.

12. The method of any one of claims 1 to 11, wherein the two surfaces are made of glass, quartz, silicon wafers, polyolefin (preferably cyclic olefin copolymer (COC) or cycloolefin polymers (COP)), plasma-activated polymers, and / or metal oxides (preferably Ta2Os, indium tin oxide (ITO), TiO2, ZrO2).

13. The method of any one of claims 1 to 12, wherein the two surfaces are microscopy slides, preferably glass microscopy slides.

14. The method of any one of claims 1 to 13, wherein the two surfaces have a size of 24 mm ± 50% x 50 mm ± 50%, a thickness of 0.170 mm ± 50%, and / or a weight of 0.605 ± 50% gram.

15. The method of any one of claims 1 to 14, wherein the cells are within a medium and preferably the medium comprises objects, preferably beads, wherein the objects allow for determining the degree of flattening of the cells by keeping the two microscopy slides apart from each other at a desired distance.

16. The method of any one of claims 1 to 15, wherein the biocompatible polymeric brushes of the antifouling coating are(a) linear brushes comprising polyethylene of glycol (PEG), lubricin, polyoxazolines, polyglycerol, dendrons, polyvinylpyrrolidone (PVP), polysaccharides, polypeptoids, polyacrylamide and / or zwitterionic polymers; and / or(b) bottlebrushes comprising polyvinylpyrrolidone (PVP), poly[(oligoethylene glycol) methyl ether methacrylate] (POEGMA), a member of the polyoxazoline family, poly(2-methyl-2-oxazoline) (PMOXA), and / or thiol end-capped poly(methacrylic acid)- g-poly(2-methyl-2-oxazoline) (PMAA-g-PMOXA).

17. The method of any one of claims 1 to 16, wherein the antifouling coating that comprises biocompatible polymeric brushes is a co-polymer having a poly(L-lysine) backbone and poly (ethylene glycol) brushes.

18. The method of claim 17, wherein the poly(L-lysine) forming the backbone has a molecular weight of 20kDa ± 50% and / or the poly (ethylene glycol) brushes have a molecular weight in the range of 2kDa ± 50% and 5kDa ± 50%.

19. The method of claim 18, wherein the copolymer comprises or has the formula:

20. The method of any one of claims 1 to 19, further comprising prior to (a):(1) activating the two surfaces in oxygen plasma, preferably for 3 min ± 50% at 0.5 mbar ± 50%;(2) coating the activated two surfaces with the antifouling agent, preferably the co- polymer of poly(L-lysine) and poly (ethylene glycol), wherein the co-polymer preferably is used at a concentration 0.1 mg / ml± 50% in a buffer, wherein the buffer is preferably 10 mM ± 50% HEPES; and(3) optionally drying the coated two surfaces, preferably in a ventilated hood.

21. The method of claim 20, further comprising prior to (1) the sterilization of the two surfaces, preferably by UV light.

22. The method of any one of claims 1 to 21, wherein the macromolecules are proteins, peptides, nucleic acids molecules, carbohydrates, lipids, nanogels, macrocycles or a combination thereof.

23. The method of any one of claims 1 to 22, wherein the macromolecules are particles.

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