FIELD OF THE INVENTION

This invention pertains to the field of bioanalytical equipment and consumables, more specifically laboratory consumables for biological and biochemical studies with a tunable functionality of both active and passive character. This invention also relates to nanostructured polymer films with different surface properties that can be used as substrate material for tissue specific 2D and 3D cell culture systems and protein production applications. More specifically this invention pertains to transparent nanostructured polymers e.g. latex films that can be functionalized with electronics, active pharmaceutical ingredients (API), antibodies, chemical agents, peptides and biomaterials for monitoring and manipulating cellular processes in either 2D or 3D cell culture systems including screening and diagnostic platforms, microfluidics, organ-on-a-chip, lab-on-a-chip with tailored channels and surface materials without geometrical limitations. The aim of the invention is to substitute the hard, flat, artificial and non-biodegradable traditional plastic cell culture materials with a biocompatible, biodegradable, easily processable, scalable and highly tunable platform for both 2D and 3D cell culture.

BACKGROUND OF THE INVENTION

Different cell types prefer different environments typical of their specific niche; e.g. soft tissues such as brain, bone marrow, and fat bear little mechanical stress and therefore prefer soft growth substrates, while stiff tissues like muscle, cartilage, and bone can sustain high levels of stress and therefore prefer harder materials. Traditionally 2D in vitro cell cultures are mostly carried out with either non-ordered plastic wells or amorphous glass cover slips and plates that degrades slowly in nature. These materials give little variation on, and control over, surface topography and are not comparable to the niches of the studied cells. Additionally, non-biodegradable plastic, such as polystyrene used in cell culture systems, is a global concern as roughly 5.5 million tons of plastic lab waste is generated yearly. Thus, an environmental friendly biodegradable platform with comparable performance to that of traditional plastic cell culture systems could decrease the plastic burden on the planet. Additionally, alternative biodegradable materials that mimic tissue specific physical and chemical properties (such as surface roughness, material stiffness, as well as wetting behavior and surface chemistry) could be used in cell cultures systems and increase the biocompatibility and minimize possible artifacts related to culturing cells on flat and hard substrates. Cell culture studies on materials with differences in the aforementioned material properties have suggested systematic relationships between the stiffness and surface properties of extracellular matrix and cell growth and differentiation [1-4] As surface topography has been shown to critically influence attachment of several microbial lines. Several studies show that nanostructured features influence bacterial adhesion and cellular functions when grown on differently manufactured surfaces [1-9]. Further, adhesion of different Salmonella species has been found to be affected by the apolar component and surface roughness of the adherent material. Functionalized nanostructured latex has the ability to regulate the adhesion of bacterium S. aureus and could thus be used in development of active surfaces in order to reduce the bacterium adhesion and growth as well as alter protein expression [8,9] To utilize these cellular responses, surfaces constructed by a biocompatible nanostructured latex polymer blends have been developed to be used in live cell culture studies, where the properties of the materials, such as surface chemistry and roughness, can be tuned and functionalized to provide improved cell-substrate interactions [5-10] Further, WO 2017/032928 and US20190002179 disclose that nanostructured surfaces offer means for passive and active control of cellular processes in order to control, enhance or decrease cell proliferation, differentiation migration and viability. SUMMARY OF THE INVENTION

The aim of this invention is to overcome the above-discussed limitations found in the cell culture systems of the prior art. This invention describes a highly processable and functionalizable two- and three-dimensional in vitro screening and diagnostic platform, both in terms of passive surface chemistry and roughness, as well as its active components such as functionalized electrodes, active pharmaceutical ingredients, antibodies and peptides. The invention is based upon a use of base (planar) surface material, where a hierarchically structured polymer is coated upon or made to be self-supporting.

Accordingly, an object of the present invention is to provide means for overcoming the problem of inefficient and/or high-cost culture systems and devices. The objects of the invention are achieved by specific cell culture devices, methods and uses, which are characterized by what is stated in the appended independent claims. Some of the specific embodiments of the invention are disclosed in the appended dependent claims. In an aspect, the present invention provides a cell culture device comprising a base and a frame structure, said base forming a bottom for a plurality of wells and said frame structure forming walls for said wells, said bottom and walls defining a volume for said wells, wherein said base comprises a first polymer material being a transparent or semi- transparent polymer film providing said bottom a surface that is bio compatible with living cells i.e. prokaryote and/or eukaryote to be cultured in said wells.

In another aspect, the present invention provides a method for preparing a cell culture device comprising a plurality of wells, said device comprising a transparent or semi- transparent polymer film providing a surface that is biocompatible with living cells to be cultured in said wells, the method comprising the steps of: a) coating a solid support with a first polymer; b) drying and sintering the polymer film formed in step a); c) optionally peeling the polymer film from the support; d) printing a second polymer on said polymer film obtained from step b) or c) by using 3D printing technology to produce a frame structure so that said frame structure provides walls for said plurality of wells or alternatively attaching a ready frame structure on said polymer film.

The first polymer film used in the method of the present invention can be, but is not limited to, e.g., latex polymer blendswith hierarchically structured topography and tuneable physico-chemical properties and roughness parameters that fulfill specific environmental demands for different cell types, thus increasing (or decreasing) the cell-material biocompatibility in the described cell culture platform. The base produced with the first polymer can be further processed to produce traditional multiwell plate geometry by fabricating a frame structure to create the wells whereto the liquid and cells may be added. The benefits of this modular design are that it enables various forms without geometrical limitations and could thus be used for 3D cell cultures. The platform can even be made to mimic tissues and organs with different materials inside the device for creating a more in vi vo- like platform thus creating e.g. 3D organoids, spheroids or organ-on-a-chip culture systems. Different blends of latices can be used as the substrate polymer that allows modification of the physical and chemical properties of the produced material in a controllable way. Thus, cell morphology, motility and differentiation, which all depend on and response to soft and stiff extracellular matrix elasticity that can be regulated tissue-specifically. In the present invention, these tissue-specific substrates can be used as coating materials for both 2D and 3D cell culture systems.

The engineered softer polymer matrix could mimic the compliance of different soft tissues to be used in growing, e.g., neuronal cells or fat cells in the described 2D or 3D cell culture system. This increases the biocompatibility of these cell-lines originating from soft tissues with cell culture materials that correspond to their environmental niche regeneration of that specific cell type. On the other hand, moderately stiff polymer blends, reminiscent of muscle, can be used in the described cell culture systems that study myogenesis (muscle cell growth and differentiation). In addition, it is possible to develop a firm substrate that would be optimized for cultivating cells that that requires stiff substrates e.g. osteogenesis (bone cell growth and differentiation).

Different template materials can be used for creating various 3D structures within the surface coating, high variations and gradients on the surface material. Additionally, different polymer blends and heat-treatments give rise to different topography and surface chemistry (both in terms of wetting and surface energy - including specific tuning of total, dispersive and polar components thereof) to match each cell and tissue specific environmental demands and needs.

The used polymeric films are intrinsically highly transparent which is desirable when designing cell culture platforms that are studied under microscopes and other instruments that use light for detecting the specimens within the platform. These polymer films can be self-supported, or then supported by, for instance, glass cover slips, plastic, polymer films or paper of different grades (Figures 1 and 7). A bimodal nanostructured surface topography can be obtained from different high throughput processes, such as inkjet printing the polymer, e.g., a latex blend on support materials such as paper, plastic, polymer or glass (Figure 8), or similarly coating the supports using rod or curtain coating methods or even different casting methods. Considerable advantages are obtained with the present invention in comparison to current standard in vitro cell culture materials. By creating different mechanical and surface topography polymer based blends, as described here, it is possible to create inexpensive biocompatible materials that can be easily optimized for a specific cell type, e.g. soft polymer for fat cells and brain cells, whereas harder polymers for muscles cells and bone cells. Currently in vitro cell cultures that mimic the in vivo environment are expensive protein based coatings and/or extracellular matrix (ECM) mimicking materials where the compositions are inconsistent and may contain unwanted components e.g. growth factors. The materials used in 3D culture, such as matrigel, collagen, fibronectin or nanocellulose gels, give a variable surface topography and stiffness, and could reduce transparency leading to observational variations. This invention enables the creation and control of the manufacturing of the polymer material so that the composition and topography is consistent giving reliable results. Preferably, the polymer used for the base could either be coated on non-functionalized polystyrene or polypropylene plastics, coated on glass and metals or on biodegradable materials such as polylactic acid (PLA) and cellulose based materials or be created as a self-supporting polymer that can be used in the presented screening system for both 2D and 3D cell culture systems.

In another aspect, the invention is directed to a use of a transparent or semi-transparent polymer film for manufacturing a microtiter plate or a multiwell culture plate.

In a further aspect, the invention is directed to a use of the cell culture device as defined above for culturing a plurality of cells and performing a biological assay on said cells.

The invented cell culture and diagnostic device can be used as a screening platform. Preferably, it can be further functionalized by, e.g., electrodes for sensing the metabolic state of the cells on each well and/or antibodies coated on the surface for an ELISA type assay. Active pharmaceutical ingredients (API) can be directly printed or coated on the screening platform and thus minimizing human errors and limiting the possible exposure of harmful substances to the end-user. The screening platform can be a micro-pattern assay where each well would have several markers for detecting for example cancer markers as a high-throughput method or a more traditional multiwell plate such as a microtiter plate, preferably comprising 6, 12, 24, 48, 96, or 384 wells. The described screening platform is modular as the base could be coupled with a frame structure to produce similar wells as a traditional multiwell plate with the additional features mentioned above. The screening platform may also comprise a lid (see Figure 1). The platform can be further functionalizable by adding antibodies or viral fragments for detecting pathogens e.g. bacterium, parasites andfor viruses, and also for identifying immunological differences of individual human leucocyte antigen (HLA) susceptibility or protection to infectious agents without expensive genetic screenings. This is achieved by functionalizing the nanostructured coating with gold electrodes and/or by direct adhesion or adding a binding layer for anchoring the streptavidin-conjugated HLA-Peptide complex, making it possible to create a ready-made diagnostic platform with simultaneous imaging and sensing of immunological processes with minimal end-user variations (Figure 10). The manufactured microtiter plate, cell culture plate or a multiwell plate with a nanostructured polymer surface can be tailored for fermentation applications such as recombinant protein production using genetically modified bacterial colonies by controlling cell growth, bio film formation and protein expression of the cultures in order to increase the protein yield and reduce production time [8-12], The designed screening platform could also be used as a microfluidics system with embedded channels coated with nanostructured polymers for fluids and cellular mobility testing combined with the possibility of using extracellular matrix (ECM) mimicking materials for creating a 3D cell culture system that could be used for organ-on-a-chip, lab- on-a-chip and/or sandwich culture systems without geometrical limitations. The measurements (readout) for the separate applications could be done with e.g. traditional microplate readers, microscopes, fluorescence instrumentations, gel and membrane imaging systems and Raman spectroscopy (SERS) [11], The readout could also be performed by first acquiring an image of the sample using a commercial office scanner, smart phone or a separate developed device. The acquired data could be analyzed with stand-alone software for giving a low-cost, fast and robust screening assay without the need of expansive instrumentation. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. A schematic presentation of the different in vitro screening platforms. The first version depicted on the top demonstrates a planar 2D screening stripe suitable for modular low-cost assays that can be manufactured by printing, coated or by roll-to-roll processing on transparent or non-transparent support. The first layer of the 2D screening stripe would be the support material that could be plastic, PLA, cellulose or self-supporting polymer. The next layer of the stripe would be the nanostructured polymer e.g. latex blend that functions as a growth substrate for each specific cell type. Then a hydrophobic layer e.g. wax or polydimethylsiloxane (PDMS) would be deposited on the stripe for creating a cell culture area that restricts the aqueous solution deposited on the stripe (cell media) much like the plastic barriers of a traditional multiwell plates creating separate wells. The disclosed screening platform can preferably be further functionalized by 1) electrodes for sensing the metabolic state of the cells on each well and/or 2) antibodies coated on the surface for an ELISA type assay. Also the 3) active pharmaceutical ingredients (API) can be directly printed or coated on the screening platform and thus minimizing human errors and limiting the possible exposure of harmful substances to the end-user. The screening platform could also be used as a 4) micro-pattern assay where each well would have several markers for detecting for example cancer markers as a high-throughput method or a non- functionalized surface for a more traditional multiwell plate. The described screening platform is modular as the base stripe could be coupled with a frame to produce similar wells as a traditional multiwell plate with the additional features mentioned above. The designed screening platform could also be used as a microfluidics system with embedded channels for fluids combined with the possibility of using extracellular matrix (ECM) mimicking materials for creating a 3D cell culture system that could be used for e.g. organ- on-a-chip, lab-on-a-chip systems.

Figure 2. Schematic illustration of atomic force microscope topographical images of latex film surface with progressively changing surface textures forming a hierarchical morphology. Hierarchical morphology describes a surface construct having a structure consisting of multiple levels, with a larger length scale features (bottom image) directly superimposed by the smaller length-scale features above them. These smaller features are in turn superimposed by even smaller length-scale features above them, continuing on the smallest length scale features possible, e.g. from micro- to nanoscale thus creating different surface properties that could be tailored for each specific cell type.

Figure 3. Example images of atomic force microscope (AFM) topographical images of different materials that can be used as cell culture substrate. To the left is depicted commercial cell culture plate made of plastic (polystyrene), to the right is a polymer blend made of dispersion of polystyrene (PS) particles (HPY83, Styron Europe GmbH, CH) and another dispersion of acrylonitrile butadiene styrene (ABS) copolymer particles (HPC26, DOW Europe GmbH, CH) that can be used to produce the required nanostructured surface.

Figure 4. Crystal violet (CV) staining showing cell viability on different cell substrates in vitro. Human dermal fibroblast seeded by a density of 50 000 cells in each well and let to grow for 96 hours. Absorbance values from each sample were normalized to the average absorbance value of the glass coverslips giving the relative of cell viability compared to a well-known substrate common for all parallel studies, which in this study was glass. Collagen, gelatin and poly-lysine was tested as an in vivo like material because they all exists in the human body and is used as coating materials for cell biological applications. Poly-lysine coated coverslip showed decreased viability compared to control sample (glass). Results shows that heat-treated latex blends in these ratios; HPY83:HPC26 (30:70), (50:50) and (80:20) significantly enhanced cell growth compared to glass coverslip and Cellstar® traditional cell culture wells. The pristine, i.e. non-IR-treated, latex blend does not increase the cell viability compared to glass and provides evidence that it is preferable to heat treat the latex blend to achieve proper surface properties. The gold (Au) coated latex blend shows no significant difference in terms of viability compared to control glass sample indicating that gold can be used as material for producing electrodes on the surface. Error bars represent ± SEM (n>3), two stars symbolizes significance level of 0.01 (P < 0.01), three stars indicate a significance level of 0.001 (P < 0.001) and four stars symbolize significance level of 0.0001 (P < 0.0001).

Figure 5. Light microscope (CelllQ instrument) images of different material compositions with cells growing on top of the material up to 96 hours. Images shows that latex films of HPY83:HPC26 blends with 30:70 wt%, 50:50 wt% and 60:40 wt% ratios enhances cell growth whereas the poly-lysine coated surface significantly decreased cell growth compared to that of control material (glass). The extracellular matrix (ECM) mimicking materials coated on the glass surface, collagen and gelatin have a significantly lower cell growth compared to the developed HPY83:HPC26 50:50 blend.

Figure 6. Human dermal fibroblasts grown on either glass coverslip or printed latex polymer blend a) Light microscopy image of HDF cells grown for 24 hours on glass substrate and after b) 96 hours showing increasing cell population c) Microcopy image of HDF cells grown on printed 50:50 PS:ABS polymer surface after 24 hours and after d) 96 hours showing increased cell proliferation compared to that of glass after similar duration e) Normalized cell viability by crystal violet staining shows an increasing trend of cell growth on the printed latex polymer after 24 hours compared to glass. Error bars represent ± SEM (n=4).

Figure 7. a) Light microscopy image of human dermal fibroblast grown on latex film with electrodes for impedance studies after 5 hours incubation b) Transparent latex coated glass coverslip with gold electrode after 72 hours incubation with HDF cells stained with crystal violet showing even distribution of cells c) Non-transparent paper stripe coated with latex and functionalized with gold electrodes incubated with HDF cells for 72 hours and stained with crystal violet. The results show that the gold electrode functionalized latex surface is biocompatible and not toxic to live cells.

Figure 8. Electrical Impedance spectroscopy (EIS) measurements performed on live human dermal fibroblast culture. Three independent experiments (samples 1, 2 and 3) show how the adherence and growth of these HDF cells affects the measured capacitance.

Figure 9. Fabricated planar 2D cell culture system with wax printed hydrophobic regions with increasing amount of seeded human dermal fibroblasts stained with crystal violet. To the right is a gold electrode functionalized planar 2D screening system that could be used for simultaneous measurements of live cells detecting metabolites, cell viability, migration and invasion assays etc.

Figure 10. The envisioned paper-based immunologic diagnostic platform. The first layer is the support material, e.g. paper, with nanostructured polymer coating for both barrier properties and cell growth interface. Functional layers include a sensing electrode and recombinant protein, e.g. HLA-epitope bound to streptavidin for anchoring to the surface, enables selective binding of immune cells to the complex. The adhered immune cells would then produce and secrete antibodies (B-cells) or cytokines (T-cells) that can be stimulated and used for a second round of testing, whereas the bound immune cell would be fixed and stained for detection and quantified using an in-house developed imaging software for detection of individual immunogenic differences in novel pathogens and vaccine epitopes.

Figure 11. Proto-model showing how 3D-printed wells of PLA can be made as a multiwell plate. The base material is paperboard coated with nanostructured latex polymer for a beneficial cell growth interface and barrier properties. The wells sustain liquids for over 72 hours without leakage the latex polymer seal.

DETAILED DESCRIPTION OF EMBODIMENTS

The term "polymer" is used herein in a broad sense and refers to materials or compounds characterized by repeating moieties or units.

The abbreviation T _g refers herein to glass transition temperature, which is a temperature range where a thermosetting polymer changes from a hard, rigid or “glassy” state to a more pliable, compliant or “rubbery” state.

The term ’’biocompatible” refers herein to the ability of a material to support cell cultures and provide suitable cell culturing conditions as well as to the ability of a material to evoke a desired response from the cultured cells.

The present invention is directed to a cell culture device comprising a base and a frame structure, said base forming a bottom for a plurality of wells and said frame structure form ing walls for said wells, said bottom and walls defining a volume for said wells, wherein said base comprises a first polymer material being a transparent or semi-transparent poly mer film providing said bottom a surface that is biocompatible with living cells to be cul tured in said wells.

A dispersion of said first polymer is used in this invention to create the biocompatible nanostructured bottom (base) substrate. The dispersion consists of one or a combination of several synthetic or naturally occurring stable aqueous dispersions or emulsions of polymer particles, such as one or more latex blends containing e.g. styrene, lactate acids, acrylic and/or butadiene groups. The polymer blends that are used for producing one level of the hierarchically structured substrate is preferably a mixture of two or more latex emulsions or dispersions with different T _g (glass transition temperature). Hierarchical morphology describes a surface construct having a structure consisting of multiple levels, with larger length scale features directly superimposed on the smaller length-scale features above them, which in turn are superimposed on the smaller length-scale features above them, continuing to the smallest length scale features possible, e.g. from micro- to nanoscale (Figure 2). In the present invention, these different structures stem from micro-scale waviness of the surfaces of the latex coating and/or the fibrous paper-based support down to the nano-scale structures and surface geometry (roughness and topology) made up by the latex particles (whether heat-treated or not) and possible depositions of even smaller scale functionalizations such as thin films or particle depositions (e.g. gold nanoparticles). The hierarchical structures of the polymeric surface coatings used in the invention are intended to mimic the extracellular matrix of each tissue specific environments so that the cell-material interaction would be closer to the in vivo situation increasing the materials biocompatibility, and help the cellular well-being at the surface (e.g. by stimulating attachment and proliferation as desired).

The thickness of the present polymer film coatings varies, however, usually in the range of 1-10 pm in thickness such that the film is transparent or semi-transparent. Said nanostructured surface can be formed by a heat treatment, e.g. by IR-heat treatment or oven, so that the hard structure-giving (high T _g ) component partly melts.

The film made of said first polymer preferably comprises a blend of, or can constitute of one or more, but typically two or more, polymeric components of different T _g . These materials can be of a stiffness (obtained, e.g., by controlled cross-linking of the used base monomers) specifically chosen to suit the study of a particular cell type (i.e. soft materials for soft tissue studies and vice versa, etc.) Preferably, the two latices comprise of polymers selected from the group consisting of: styrenes, acrylics, butadienes (i.e. 1,3 -butadiene), lactates and copolymers thereof. Most preferably, said two latexes are styrene and/or acrylic based polymers. Said two latexes are preferably mixed about 30:70, 40:60, 50:50, 60:40 or 70:30. The preferable particle size for the higher T _g component is 100-300 nm and can provide barrier properties, mechanical strength and integrity for the film.

Most preferably, said two latexes are polystyrene (PS) and styrene butadiene acrylonitrile (ABS) copolymer. Said two latexes are mixed in a desired ratio to obtain the desired roughness, which can be used to enhance cell proliferation and/or cell adhesion. The molar ratio (or alternatively wt%:wt%) of PS: ABS latexes in the blend, is dependent of the application, and can be in the range of about 10:90 - 90:10, preferably about 20:80 - 80:20, more preferably about 30:70 - 70:30, 30:70 - 80:20, or 20:80 - 70:30, even more preferably about 40:60 - 60:40.

The invention utilizes the described support material for creating a screening platform either in a 2D or a 3D format suitable for supporting cell cultures compatible with flexible electronics or electrodes, antibodies for detecting proteins of interests, API for testing and screening purposes as well as diagnostic applications, with tuneable surface properties (surface chemistry and wetting) and stiffness for detecting cell growth and cell adhesion, micro-patterned assays.

Thus, typically, the substrate used in the screening platform comprises a support layer of which is either non-transparent or, preferably, transparent. The base support material extends preferably along a 2D plane, in the case of transparent support the material should allow for transmission of light, both light in the visible range and fluorescent range, through the structure using but not limited to about 90° against the substrate plane for detecting the sample of interest e.g. cells, proteins or materials.

The non-transparent support can preferably be made of e.g. paper, cardboard, glass, metal or polymer. For the purposes of these embodiments the film with the support material is considered non-transparent when the transmission of light in visible range 50 % or lower.

The transparent support can be made of e.g. plastic, glass, PLA, a paper- or wood- based material, glass, plastic or polymer material of a preferably biodegradable character. Examples of transparent supports are glass coverslips and films of polystyrene (PS) or PLA. Preferably, the film is transparent or semi-transparent with the transmission of light in visible range being over 50 %, more preferably in the range of 70-90% or 70-99%.

The polymer film can be further, or alternatively, functionalized, using e.g. printing techniques or roll-to-roll processing, with electronic devices or electrodes, coatings of any kind, API, chemicals, biochemicals including antibodies or proteins of interest on the nanostructured base film.

The term “printing” refers herein to the process of either producing the polymer film itself or functionalization of the polymer film using any printable devises with electronics, applying API on the surface or chemicals, or adhering antibodies or proteins of interest on the film using any printable device commercial or tailored. The term “3D printing” refers herein generally to additive manufacturing and to the process of either producing the three-dimensional structures of polymer film itself or functionalization or any other polymer for creating a three-dimensional cell culture platform. The term "3D printing" summarizes a large variety of technologies, but at its core, 3D printing is generally a process in which a three-dimensional structure is formed by the cumulative fusion of discreet particles (such as plastics and metals) layer by layer. An example of a common technique for layering molten plastic to form 3D products is fused deposition modelling (FDM), which is also known as fused filament fabrication (FFF). This technique allows one or more plastic materials to be heated and deposited for cooling to form a 3D product.

In preferred embodiments, said frame structure of the cell culture device is obtained by 3D printing of a second polymer material on said base of the cell culture device, said second polymer material being different from said first polymer material. More preferably, said second polymer is a thermoplastic polymer such as acrylonitrile butadiene styrene (ABS) or polylactic acid (PLA). Other materials such as polycarbonates (PC), polyamides (PA) and polystyrenes (PS) are also available, and more recently, polyether ether ketones (PEEK). Alternatively, said frame structure is by molding or gluing a ready frame structure on said base, wherein the material of said ready frame structure is preferably different from said first polymer material. The material of said ready frame structure is preferably selected from a group consisting of plastics, natural based materials such as cellulose and textiles.

Electrically and electrochemically active layers for electric modulation and sensing can be deposited or printed on the polymer film. For example, ultra-thin and conductive gold elec trodes (UTGE) with 50% transmission can be evaporated onto the latex surface and used in the screening platform for detecting electrochemical signals from the cells (Figure 1, 7, and 8). The sensing electrodes allows continuous monitoring of cellular metabolites and mole cules in the aqueous solution in the sample of interest during cell adherence, growth, mi gration and death. Alternatively, the electrodes can also be used as such for measuring the resistance and/or conductance of the sample e.g. cells interacting with the substrate materi- al that can be functionalized with antibodies, proteins, API, chemicals, etc. (Figure 1). Preferably, the semi-transparent electrode is an ultrathin metal film electrode (UTMF), such as an ultrathin gold film (UTGF) electrode, or a conductive semitransparent or trans parent polymer such as PEDOT:PSS. The described modular screening platform (Figure 1) enables simultaneous high-resolution optical imaging, high-throughput assays with electrical measurements opening, thus opening a new dimension of detectable variables in cell culture systems such as cell adherence, cell growth and migration and further, without losing any possibilities of older technologies for cell culture (i.e. plastic, non-functionalized well plates). Specific advantages of electrical methods are the ability to detect low concentrations of biological analytes e.g. cellular metabolites, biomolecules, biomarkers such as cancer markers, hormones without the need of labeling, fixing or staining the sensitive biological sample.

One typical platform is a planar 2D cell culture system made to be non-transparent or transparent modular multiwell platform (stripe) where the support material would preferably be biodegradable material e.g. plastic, PLA, cellulose based materials. With a nanostructured polymer-based surface topography for enhanced cell-material interactions, tailored surface chemistry and wetting, and electrodes for low-cost, robust, stand-alone analysis is possible using visible light, absorbance, fluorescence, luminescence techniques, and electrical measurements in real-time giving simultaneously detailed information of different variables regarding status of the sample, e.g. cell adherence, growth, migration, metabolites and immune cell attachment and antibody production (Figure 10). These measurements could be done with however not restricted to traditional microplate readers, microscopes, fluorescence instrumentations and Raman spectroscopy (SERS). The readout could also be performed by first acquiring an image of the sample using a commercial office scanner or smart phone, in the case of more simple optical studies. Quantified could be done using an analytical tool capable of giving an estimate of the staining intensity of particular staining agent, e.g. crystal violet for estimating cell viability of the cell-material interaction compared to a known control surface where cells grow on. The planar 2D platform would have regions of interest (ROI) limited by a wetting controlling material (e.g. hydrophobic polydimethylsiloxane (PDMS) or waxes, etc.) colored with a dye for easy visual identification (preferably non-toxic, e.g. food colorants) to discriminate the region of interest (ROI). The ROI would then be the un-dyed circular area(s) where the nanostructured polymer coating is in contact with the sample, i.e. where the cells would grow (Figure 1; left). The ROI would harbor the aqueous solution (cells, medium, proteins etc.) on top of the polymer coating. One of the greatest advantages of the planar screening platform is the compact and potential stand-alone usage enabling consumer products to be developed without geometrical limitations. Another version of the platform would be a 2D cell culture system that could be built to a multiwell screening platform that would include but not limited to be manufactured ac- cording to the ANSISLAS-Standards 1 to 4 - 2004. Enabling the use of the invented screening platform with current instrumentations directly with minimal optimization nec- essary (Figure 1; middle). The major benefits of this screening platform would be the tai- lored surface chemistry that could mimic cell specific demands to a “2.5D” structure that would resemble the tissue of living materials produced by inexpensive polymers e.g. latex. Another major benefit of the listen platform is the functionalization possibilities as the pla- nar base explained in more details above that can easily be coated or printed with electron- ic, API and bio molecules for diverse in vitro assays. The frame that would be joined/fused/glued or preferably printed to the base could be made by plastic, PLA, cellu- lose or other polymers that enables diverse geometrical wells that can be functionalised or made up by heterogenous or polymorphous materials - not only the traditional cylindrical well structures. With ‘functionalized materials’ is here intended materials with a coating, nanostructure or varying physical or chemical properties as described earlier in the text. With ‘heterogenous materials’ is here intended materials that can, in the well make of re gions of different character, both in the physical and chemical sense. That is the well walls could have, for instance, conductive and non-conductive areas, porous and non-porous areas, bioretentive or -supportive areas. In a preferred embodiment, the height of said walls is at least 0.5, 0.8, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mm, when measured from the surface of the bottom of a well.

In preferred embodiments, said frame structure of the cell culture device comprises a diffu- sion channel, such as a microchannel, or a membrane which provides a diffusion channel or a membrane between at least one of the plurality of wells and an adjacent well. More preferably, said membrane is a porous membrane excluding molecules of a specific size or diffusion rate from passing through said channel.

The most advanced version of the invention described herein is the 3D cell culture and or screening platform capable of doing simultaneously the above-mentioned assays and measurements with additional micro fluidics, lab-on-a-chip and organ-on-a-chip capabilities (Figure 1; right), 3D electrodes for monitoring and manipulating cellular processes for creating and advanced controllable platform. The platform could be 3D printed/fabricated to each specific organ of interest with ECM materials that would match each tissue specific environmental demands. Together with microfluidic channels that allow changing of the medium or to introduce other cells to the system, the platform may comprise additional membranes for selectively allowing molecules, hormones, treatments or API to cross between different compartments monitored in real-time. The screening platform could be produced as a ready-made modular assay so that the end user only has to add the cells whereas the assay contained the ECM mimicking material with additional electrodes, API, biomarkers, base substrate etc. The intended screening platform could be produced as a sandwich model where different material compositions would be stacked on top of each other mimicking for example the invasion of cells from one tissue to another or penetration of different membranes. Another benefit is having 3D layers of different materials, API, or sensing electrodes is that the cells could be manipulated and controlled at different spatial and temporal arrangements.

In further embodiments, the present invention is thus directed to a method for preparing a cell culture device comprising a plurality of wells, said device comprising a transparent or semi-transparent polymer film providing a surface that is biocompatible with living cells to be cultured in said wells, the method comprising the steps of: a) coating a solid support with a first polymer; b) drying and sintering the polymer film formed in step a); c) optionally peeling the polymer film from the support; d) printing a second polymer on said polymer film obtained from step b) or c) by using 3D printing technology to produce a frame structure so that said frame structure provides walls for said plurality of wells or alternatively attaching a ready frame structure on said polymer film.

In a preferred embodiment, said method comprises a further step of functionalizing the polymer film obtained from step b) or c) by coating or printing on said film one or several of the following: peptides or proteins such as antibodies, chemical agents, active pharma- ceutical ingredients (API), or electrodes.

In another preferred embodiment, said method comprises a further step of functionalizing the polymer film obtained from step b) or c) by coating or printing on said film a thiol monolayer, a semi-transparent electrode or a combination thereof. It will be obvious to a person skilled in the art that, as the technology advances, the in- ventive concept can be implemented in various ways. The invention and its embodiments are not limited to the examples described below but may vary within the scope of the claims.

EXPERIMENTAL SECTION

Materials and Methods

The surface manufacturing process

Coating phase

The desired polymer blend e.g. latex dispersion (emulsion) is applied on a supporting substrate surface of choice by e.g. spin coating, manual drop deposition or roll-to-roll coating methods like rod coating or reverse gravure coating or by printing techniques. The thickness of the obtained latex layer, having a preferential layer thickness of 1-10 pm, is such that the film is transparent or semi-transparent with the transmission of light being over 50%. The supporting substrate can be transparent, semi-transparent or non-transparent material, such as silicon-based (e.g. glass, Si02), cellulose based material such as paper or cardboard, metallic, glass, plastic, or polymeric (e.g. PDMS, PLA) or any combination of them. The surface of the supporting substrate can be pre-structured with macro-, micro and/or nanoscale features to produce the hierarchical morphologies (Figure 2). This pre- structuring can be made e.g. by lithography, laser-cutting, template-stripping, or 2D and 3D printing.

Drying and sintering phase

The drying and sintering phase involves the modification of the polymer blend e.g. latex film surface by thermal radiation using e.g. an infrared dryer to form micro- to nanoscale surface features (Figure 2). The density of surface features on the latex film can be controlled by the changing e.g. the ratio of different components in the blend. Thus hierarchical morphologies of higher order can be superimposed on the pre-structured supporting substrate. After the drying and sintering phase, the polymer film on supporting substrate can be applied directly to cell assays or, alternatively, the polymer film can be peeled off from the supporting substrate by the help of e.g. sonic agitation and thus forming self-supporting polymer films to be applied as the base layer in the in cell assays described in Figure 1.

Functionalization phase

The polymer films e.g. latices (on a supporting substrate or as a self-supporting film) can be further functionalized by, e.g. metal films, cell adhesive proteins, API, peptides, fragments of pathogens, antibodies, biomarkers, insulating, semi-conducting or conducting polymer material, carbon-based material, metallic and silicon-based insulating, semiconducting or conducting nanoparticles, gradients of the nanostructured material blends, or any combination of them as the latex surface offers excellent adhesion towards materials deposited on it (Figure 1). Deposition of the functionalized layers can be done e.g. by physical vapor deposition, atomic layer deposition, printing, self-assembly, manual drop deposition, coating etc. If the thickness of functional layer is small enough (<10 nm), sufficient transparency can be still obtained (Figure 8). For example, transparent electrodes enabling electric or electrochemical monitoring or sensoring during cell assay can be fabricated on transparent polymer films placed on a transparent supporting substrate or as self-supporting films by evaporating ultrathin gold films on the latex surface.

Characterization of the polymer films

Atomic force microscopy (AFM) was used for mapping of surface mechanical properties such as stiffness and modulus. In addition, force mapping of adhesive tip-sample interactions provides information about chemical heterogeneity of the surfaces, spatial distribution of components and even phase separation. The tip-sample thermodynamic work of adhesion of e.g. composite materials yields information about the local surface energy levels. AFM is furthermore used for characterizing the prepared latex surfaces before and after exposure to cells and ECM.

3D printing of well frame

Fabrication of 3D multi- well test arrays using 3D printer KIMM SPS1000 Bioplotter. In this setup, a paperboard base was first coated with the nanostructured latex film onto which polylactic acid (PLA) or a lignin/PLA mixture was 3D-printed, forming the walls of the wells in the analytical test array (Fig. 11). The adhesion between the well walls and the base material was good, even without using any added adhesives, e.g. glues. The adhesion of the well walls onto the surface is based on thermal fusing during the printing, which occurs at temperatures above the latices’ glass transition temperature. This process, since working without any extra additives, is a real advantage minimizing any unexpected issues, toxicity and responses in cell viability and material compatibility. The wells held a food dye solution for 72 hours without visible capillary leakage or penetration, demonstrating the potential of the proposed screening platform.

Cell culture

Human dermal fibroblasts (HDF) or cervical cancer cells (HeLa) were obtained from ATCC (Manassas, VA, USA) and maintained in DMEM medium (Sigma- Aldrich, USA) supplemented with 10% foetal calf serum (BioClear, Wiltshire, UK), 2mM L-glutamin, 100 U/ml penicillin, 100 μg/ml streptomycin at 37 °C in a 5% C02 / 95% 02 and 90% relative humidity atmosphere and maintained under sterile conditions in a cell culture incubator.

Coating of extracellular matrix mimicking materials

For adding ECM mimicking materials on the glass surface the coverslip were first sterilized by incubating them in 95% ethanol and dried before coating procedure. The coverslips were placed in a multiwell plate containing sterile working solution of either: 50 Lig/ml poly-D-lysine, 2% (w/v) gelatin solution then incubated for 1 hour at 37°C and dried before use. The collagen was coated by incubating the coverslips overnight at 4°C using sterile rat tail type I collagen at 100 pg/ml concentration prior to use.

Cell viability analysis

For monitoring cell adhesion, morphology and cell growth of the different polymer blends coated or printed together with currently used ECM mimicking materials in vitro, human dermal fibroblast (HDF) were seeded (0.5 x 10 ⁶ /well) on 24 well plates containing each substrate of interest. On the next day the plates were inserted in the CelllQ automated live cell imaging device were the cells were monitored for 72 hours with an imaging cycle of 3 hours giving a total time of 96 hours of cell growth on each substrate. In order to quantify the cell viability attached on the surface normalized to the know glass surface as the control, crystal violet was employed. In short, after 96 hours incubation cells were first washed with PBS, and then stained with 0.2 % crystal violet in 2 % ethanol at room temperature for 10 minutes. Thereafter cells were washed extensively with distilled water and let dry overnight, the crystal violet were then resolubilized with 1 % sodium dodecyl sulfate (SDS). The supernatant was then transferred to a new 24 well plate without the latex coated materials after which the absorbance was measured at 570 nm with Hidex sense microplate reader (Hidex, Turku, Finland).

Impedance measurements Electrical Impedance spectroscopy (EIS) measurements were performed using a portable electrochemical interface and impedance analyzer (CompactStat, Ivium Technologies, The Netherlands). The experiments were carried out with a two ultra-thin and conductive gold electrodes (UTGE) on either latex coated glass substrate or paper support for testing the potential use of electrochemical measurements on live cells. Human dermal fibroblast were seeded on the electrode functionalized latex surface and the impedance measurements throughout the work were recorded at a constant dc-potential (0 V) and with an applied sinusoidal excitation signal of 10 mV at a frequency range of 10000 Hz - 10 Hz.

Results and discussion

Topographical characterization of the nanostructured polymer substrate Different kinds of support materials e.g. glass, paper, plastic were used for the preparation of the latex polymer films and by changing the blends ratios of the PS and ABS different surface properties can be obtained. After an IR treatment, a distinct nanostructured topography with bimodal height distribution can be obtained, depending on the ratio of high Tg and low Tg components in the polymer blend (Figure 3). For example, the nanostructured features can be prepared by rod-coating the latex blend dispersion on a pigment coated paper substrate or by printing the latex blend on glass surfaces (Figure 6&9).

Optical characterization of the obtained film

Optical transparency of the different latex films coated materials were determined by UV/vis spectrophotometer in transmission mode. The results show that about 80-90% optical transmission in the visible light region (400-700 nm) was achieved when coating the latex polymer blend on glass substrate and even when coated with ultra-thin gold films was the transparency around 70% [5-10] Electrochemical characterization

Impedimetric measurements were carried out with the transparent nanostructured latex coated on glass coverslips for extended time periods using cell medium to test the long term stability of the UTGF electrodes. The capacitance of the UTGF electrodes remained extremely constant after the initial stabilization using only cell medium. The obtained capacitance decrease shortly after the cells where added to the sample and after the cells adhered to the surface and started to divide at around 10 hours incubation the capacitance started to rise and continued to increase thought the whole incubation up to 72 hours (Figure 7&8).

Cell-material biocomapbility

Human dermal fibroblasts (HDF) were grown on different substrates for 96 hours under real-time imaging using CelllQ instrumentation, which after cell proliferation was determined by measuring the cell population, by crystal violet staining (CV). As a reference material HDF cell were growth on glass cover slips was and compared to the other materials using statistical analysis. A commercial well plate surface, Greiner CELLSTAR®, and a smooth PDMS surface were included in the comparison as control materials. Furthermore, materials used in 3D cell cultures was included in these studies e.g. poly-lysin, collagen and gelatin. The cell growth studies shows that our developed nanostructured latex surface can influence cell growth and that the 50:50 PS:ABS blend polymer increased cell viability while keeping the optical properties (transparency) at the highest of the different blend that is most suited for cell cultures (Figure 4). Intriguingly the materials used in 3D cell cultures had lower cell growth than that of the selected 50:50 blend demonstrating that our developed nanostructured materials could be used for tissue specific cell culture studies [5-10] Additionally, the 50:50 PS:ABS polymer blend was successfully printed on a glass surface with similar nanostructured surface that enables cell growth (Figure 6).

Screening platform

In order to demonstrate that the nanostructured polymer can be used for developing a screening platform; a paper supported functionalized latex-film was produced (Figure 9). In this setup wax printing was used for controlling the wetting areas where the liquid could be suspended on. Human dermal fibroblasts and human cervical cancer (HeLa) cells were seeded with increasing amounts of cells on the paper based screening platform and both cell lines showed successful cultivations of cells seen as an increase of crystal violet staining (Figure 9) [5,10] Furthermore, the ultrathin gold film electrode can be printed on top of the latex coated paper based screening platform for adding an additional measuring dimension.

REFERENCES

Patent Literature US20190002179 WO 2017/032928 Non-Patent Literature

1. Discher DE, Janmey P, Wang YL. Tissue cells feel and respond to the stiffness of their substrate. Science. 2005 Nov 18;310(5751): 1139-43.

2. Walcott S, Sun SX. A mechanical model of actin stress fiber formation and substrate elas- ticity sensing in adherent cells. Proc Natl Acad Sci U S A. 2010 Apr 27;107(17):7757-62.

3. Flemming RG, Murphy CJ, Abrams GA, Goodman SL, Nealey PF. Effects of synthetic micro- and nano-structured surfaces on cell behavior. Biomaterials. 1999 Mar;20(6):573- 88.

4. Abagnale G, Steger M, Nguyen VH, Hersch N, Sechi A, Joussen S, Denecke B, Merkel R, Hoffmann B, Dreser A, Schnakenberg U, Gillner A, Wagner W. Surface topography en- hances differentiation of mesenchymal stem cells towards osteogenic and adipogenic line- ages. Biomaterials. 2015 Aug;61:316-26.

5. Rosqvist E, Niemela E, Venu AP, Kummala R, Ihalainen P, Toivakka M, Eriksson JE, Pel- tonen J. Human dermal fibroblast proliferation controlled by surface roughness of two- component nanostructured latex polymer coatings. Colloids Surf B Biointerfaces. 2019 Feb 1;174:136-144.

6. Bagherifard S, Hickey DJ, de Luca AC, Malheiro VN, Markaki AE, Guagliano M, Webster TJ. The influence of nanostructured features on bacterial adhesion and bone cell functions on severely shot peened 316L stainless steel. Biomaterials. 2015 Dec;73:185-97.

7. Mei L, Busscher HJ, van der Mei HC, Ren Y. Influence of surface roughness on strepto- coccal adhesion forces to composite resins. Dent Mater. 2011 Aug;27(8):770-8.

8. Juvonen H, Oja T, Maattanen A, Sarfraz J, Rosqvist E, Riihimaki TA, Toivakka M, Kulo- maa M, Vuorela P, Fallarero A, Peltonen J, Ihalainen P. Protein and bacterial interactions with nanostructured polymer coatings. Colloids SurfB Biointerfaces. 2015 Dec 1 ; 136:527- 35.

9. Juvonen HI, Maattanen A, Lauren P, Ihalainen P, Urtti A, Yliperttula M, Peltonen J. Bio- compatibility of printed paper-based arrays for 2-D cell cultures. Acta Biomater. 2013 May;9(5):6704-10.

10. Rosqvist E, Niemela E, Frisk J, Oblom H, Koppolu R, Abdelkader H, Soto Veliz D, Men- nillo M, Venu AP, Ihalainen P, Aubert M, Sandler N, Wilen CE, Toivakka M, Eriksson JE, Osterbacka R, Peltonen J. A low-cost paper-based platform for fast and reliable screening of cellular interactions with materials. J Mater Chem B. 2020 Feb 14;8(6):1146-1156.

11. Durmanov, Nikolay & Guliev, Rustam & Eremenko, Arkady & Boginskaya, Irina & Ryzhikov, Ilya & Trifonova, Ekaterina & Putlyaev, Egor & Mukhin, Aleksei & Kalnov, Sergey & Balandina, Marina & Tkachuk, Artem & Gushchin, Vladimir & Sarychev, An- drey & Lagarkov, Andrey & Rodionov, Ilya & Gabidullin, Aidar & Kurochkin, Ilya. (2017). Non-labeled selective virus detection with novel SERS-active porous silver nano- films fabricated by Electron Beam Physical Vapor Deposition. Sensors and Actuators B: Chemical.

12. Gupta SK, Shukla P. Sophisticated Cloning, Fermentation, and Purification Technologies for an Enhanced Therapeutic Protein Production: A Review. Front Pharmacol. 2017 Jul 4;8:419.