Functionalized substrate to manipulate cell function and differentiation

Abstract

To provide methods allowing control of culture conditions that is more robust, in particular less independent from existing knowledge and / or experience, as well as means that can be suitably employed in such methods.SOLUTION: The invention relates to a scaffold for steering cells in a predetermined direction of cell functionality, preferably a cell differentiation scaffold for steering cells in a predetermined direction of cell differentiation. The scaffold comprises a polydimethylsiloxane (PDMS)-, or rubber-, or silicone-based polymeric surface, and one or more cell functionality-inducing stimuli, preferably one or more cell differentiation-inducing stimuli, coupled to the polymeric surface.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates generally to the fields of biotechnology, bioinformatics and related technologies. More specifically, the present invention relates to a substrate or scaffold for steering cells in a predetermined direction of cellular function, and to a culture unit having such a substrate or scaffold. The present invention also relates to an in vitro method for generating cell cultures, tissues or organs by steering cells in a predetermined direction of cellular function. The present invention further relates to a non-transitory storage medium having stored thereon instructions executable by a digital processing device for carrying out the above-mentioned method. The present invention further relates to a computer program comprising program code means for causing the digital processing device to carry out the above-mentioned method when executed on the digital processing device. The present invention further relates to a system for carrying out the above-mentioned method. [Background technology]

[0002] Cellular function and differentiation during embryonic development, adulthood and disease are controlled and steered by approximately 15 signaling pathways, such as the Hedgehog, PI3K and estrogen receptor pathways. Pathway activity is regulated by cell-extrinsic factors, including small molecules, secreted proteins, and environmental stimuli such as temperature and oxygen, among others. These stimuli can originate from other cells within the organism or can come from the organism's environment. Within an organism, cells communicate with each other by sending and receiving secreted proteins, also known as growth factors, morphogens, cytokines or signaling molecules. Reception of these signaling molecules triggers intercellular signaling cascades that ultimately result in changes in gene transcription or expression. This process is thought to regulate the behavior of a vast number of cells.

[0003] In higher organisms, intercellular signaling pathways play a key role in coordinating and regulating cell division. They ensure that cells divide synchronously and, if necessary, stop cell division and enter a quiescent state. Cellular communication is of great importance in the differentiation and development of organisms. The development of organisms is based on genetic programs that constantly utilize intercellular and intracellular signaling pathways. Signal molecules produced by a cell affect and change the function and morphology of other cells in the organism. Intercellular signaling pathways are also important for the processing of sensory information. External stimuli, such as optical and acoustic signals, stress, nutrient gradients, etc., are registered by sensory cells and transmitted to other cells of the organism via intercellular signaling pathways. Naturally, a variety of timely and spatially controlled stimuli are provided to regulate the behavior of cells.

[0004] For example, during embryonic development, stem cells differentiate into all types of somatic cells. Stem cells proceed through multiple defined differentiation steps, eventually maturing into fully differentiated cells such as skin cells, neurons, etc. During adulthood, stem and progenitor cells reside in organ niches under very specific conditions and differentiate to replace organ cell types lost due to physiological processes (e.g., in the intestinal mucosa) or due to disease-related organ damage. In benign and malignant tumors, cells lose their differentiation function, allowing them to start dividing and metastasizing, which is also regulated by the (spatiotemporal) activity of signaling pathways. All of this is regulated by the highly regulated (spatiotemporal) activity of signaling pathways.

[0005] In other diseases, there is often aberrant activity of signal transduction pathways that play a causative role in the disease, its progression and response to treatment.

[0006] Investigation of cell and tissue function or of specific diseases to better understand (patho)physiology and develop drugs and therapies (such as regenerative medicine) requires in vitro cell and tissue culture model systems for healthy and / or diseased cells / tissues. The success of these and other in vitro applications that rely on cells to obtain specific cellular behaviors depends primarily on the immediate state of the cells in question. Therefore, physical, chemical and biological control of the cellular microenvironment is considered crucial for the ability to spatially and temporally direct and control cellular behavior in clinically relevant applications such as the generation of three-dimensional tissue engineering scaffolds.

[0007] The precise control of multiple stimuli in the cell culture microenvironment that regulate intracellular signaling and ultimately cell phenotype is particularly important in stem cell culture and differentiation (as well as other cultures). Reproducing in vivo (in vivo) cells / tissues by in vitro culture is possible, provided that the necessary stimuli (stimuli) are provided in the correct spatiotemporal manner. Stimuli may consist of ligands (mainly proteins) that bind to specific cell membrane receptors and activate said receptors, activating specific intracellular signaling pathways. Activation of such pathways leads to functional changes of the cells. Moreover, the culture surface needs to mimic the natural in vivo cell environment, which is an obstacle to optimally and most efficiently differentiate and mature stem cells in the required direction.

[0008] For already differentiated cells, it is generally more important to influence in a controlled manner the functions of the cells, such as cell division, migration, metabolism, etc. However, the same requirements apply as given for stem cell differentiation.

[0009] Achieving such precise control in vitro is difficult for conventional culture systems, but microfluidic devices are believed to provide a solution to this problem. For example, a review by Zhang et al. in Future Sci OA, 2017, 3(2), FS0187 describes microfluidic devices as ideally suited for culturing and maintaining stem cells by providing stimuli to generate in vivo-like microenvironments. Advantageously, microfluidic devices offer, among other things, well-defined surface features and patterned scaffolds, the potential to facilitate stimulation of cells with both biochemical and structural stimuli, including mechanical stress and electromagnetic forces. Furthermore, microfluidic devices can simultaneously control multiple stimuli over space and time with high precision, thereby providing an ideal and well-defined platform for stem cells. Summary of the Invention [Problem to be solved by the invention]

[0010] A drawback of known techniques, whether performed in conventional culture systems or microfluidic devices, is that they mainly depend on the control of culture conditions. The term control used by various authors, including Zhang et al., should be understood to be a broad description of the possibility to adjust culture conditions to provide a suitable microenvironment for the corresponding purpose. The selection of appropriate culture conditions is based on existing knowledge and / or extensive experience by the experimenter. According to these methods, the results are highly dependent on the level of existing knowledge and / or experience.

[0011] It would therefore be desirable to have methods that allow control of culture conditions, which are more robust and in particular less dependent on existing knowledge and / or experience, as well as tools that can be suitably used in such methods. [Means for solving the problem]

[0012] According to a first aspect of the present invention, the above problem is at least partially solved by a substrate or scaffold for steering cells in a predetermined direction of cell function, said substrate or scaffold comprising an elastomeric material to which one or more cell function inducing stimuli are attached.

[0013] Said substrate, preferably with or being a scaffold, is for directing cells in a given direction of cell function. Such directing can usually be performed during culture or culturing of cells. The substrate comprises an elastomeric material to which one or more cell function directing stimuli are bonded. Said stimuli are for supporting or causing said steering. This means that said stimuli are preferably present at least on the elastomeric surface of said elastomeric material so as to be available for such steering during culturing of cells. The presence of stimuli at the surface of the elastomeric material provides a controlled environment in which said steering can take place at or near the surface of the elastomeric material.

[0014] The base material (substrate) may be a cell differentiation substrate, or the scaffold may be a cell differentiation scaffold for directing cells in a predetermined direction of cell differentiation.

[0015] The substrate or scaffold may be part of a culture unit. There is provided a culture unit comprising a cell culture chamber having therein a substrate or scaffold according to any of the claims, the substrate or scaffold thus being arranged to be partially located within said chamber.

[0016] The culture unit may be part of a microfluidic device. Such a device may have other features useful for culturing cells. For example, one or more conduits may be implemented to transport fluids and / or gases to and from the substrate to aid in culturing cells.

[0017] The substrates, scaffolds and culture units are thus designed to provide a controlled environment for directing cells in the desired direction of cellular function during cell culture.

[0018] In accordance with the above object, there is provided the use of a substrate or scaffold according to any one of claims 1 to 8, or a culture unit according to claim 8, for generating a cell cultured tissue or organ.

[0019] Thus, a second aspect of the invention relates to a method for generating cell cultures, tissues or organs with the guidance of cells in a predetermined direction of cellular function, the method comprising: (a) determining first culture conditions that control the activity of at least one first signaling pathway of a cell to be cultured, the activity of the at least one first signaling pathway being selected to control cell behavior in a first predetermined manner; (b) culturing cells in a culture unit under the first culture conditions; Optionally, the method further comprises: (c) inferring the activity of the at least one first signaling pathway in a cell sample obtained from the cell culture; and (d) comparing the predicted activity with the selected activity; has.

[0020] The inventors of the present invention have found that, despite a steady increase in knowledge and experience regarding the stimuli required for cells to acquire certain cellular behaviors, it is difficult to consistently guide cells in the same direction. The level of knowledge and experience directly influences the results obtained. If modifications to the culture conditions are made during the culture, this is based on the deviation of the actual culture conditions from those that lead to the desired cellular behavior according to the existing knowledge and / or experience. It is not taken into account that the existing knowledge and / or experience may be insufficient or inaccurate, and therefore the culture conditions assumed to be appropriate may be suboptimal or even erroneous.

[0021] According to the present invention, a robust method is provided in which cells can be controlled to reproducibly adopt a particular cellular behavior that is less dependent on existing knowledge and / or experience as a known method. The method of the present invention allows to directly determine whether cells actually respond in an expected manner to the culture conditions determined in step (a). Thus, in a first step (a), culture conditions are determined that are known or at least assumed to induce a particular cell (i.e., cell type) towards a desired cellular behavior. In step (b), a cell culture is performed by culturing cells of the same cell type as in (a). Pathway activity is inferred in step (c). Comparing the inferred activity (activity) with the activity selected to control the cellular behavior in a first predetermined way (i.e., selected activity) of at least one first signaling pathway in the culture makes it possible to monitor the cellular response at the level of the cell culture, to monitor the progress of the cell culture to acquire a particular cellular behavior, to confirm or reject that the cell culture has acquired the desired cellular behavior, or to guide it in a desired direction, etc. More specifically, the present invention allows to draw conclusions regarding cellular behavior by quantitatively inferring the activity of the signaling pathways that preferably mediate the respective cellular behavior. For example, differentiation of stem cells into a particular tissue may involve activation of one or more pathways. By inferring the activity of said one or more pathways, it is possible to ensure that the respective signaling pathways are in fact active. Information on pathway activity, especially quantitative information, can be advantageously used for the purposes of the present invention as a basis for minimizing deviations from the desired cell behavior, thus allowing precise control of cell behavior and directing cells in the desired direction. Thus, the present invention provides a method that allows cell culture in a more controllable manner than is possible with existing methods.

[0022] According to a fourth aspect, the problem is solved by a non-transitory storage medium storing instructions executable by a digital processing device to perform the method of the invention.

[0023] According to a fifth aspect, the problem is solved by a computer program comprising program code means which, when executed on a digital processing apparatus, causes said digital processing apparatus to carry out the method of the invention.

[0024] According to a sixth aspect, the object is to provide a system for carrying out the method of the invention, (a) a culture unit or scaffold; (b) a sensor unit for inferring activity of at least one first signaling pathway; (c) a controller configured to compare the estimated activity of the first signaling pathway with a selected activity, the selected activity being selected to control a cellular behavior in a first predetermined aspect of the invention or a computer program of the invention; has.

[0025] Further advantages will become apparent to those of ordinary skill in the art upon reading and understanding the following detailed description.

[0026] This application has described several preferred embodiments. Other modifications and alterations will occur to those skilled in the art upon review and understanding of the above and following descriptions. It is intended that this application be construed as including all such modifications and alterations insofar as they come within the scope of the appended claims and their equivalents.

[0027] Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention from a study of the disclosure and the appended claims.

[0028] The substrate or scaffold of claim 1, the culture unit of claim 8, the use of claim 9, the method of claim 10, the non-transitory storage medium of claim 17, the computer program of claim 18 and the system according to claim 19 have similar and / or identical preferred embodiments or make use of corresponding features, in particular as defined in the dependent claims.

[0029] It is to be understood that a preferred embodiment of the invention can also be any combination of the dependent claims or above embodiments with the corresponding independent claims.

[0030] These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter. [Brief description of the drawings]

[0031] [Figure 1A] FIG. 1A exemplarily shows pathway activity during differentiation of human stem cells (human embryonic stem cells, undifferentiated; hESC undiff) into endoderm by SR-1 induction (SR-1 ind). Mesoderm (MD) is formed by AFBLy differentiation of hESC (AFBLy-diff hESC) (log2odd values ​​after 3 days (3d) are shown). Pathway activity was obtained by Philips pathway analysis of Affymetrix U133Plus2.0 microarray data available in the public GEO dataset GSE52658. The Phillips pathway activities measured at each differentiation step are shown as log2odds scores for the following pathway activities: PI3K-FOXO, Hedgehog (HH), TGF-β, Wnt, Notch, and STAT3 pathways, next to the annotation for each individual sample (e.g., anterior primitive streak (APS), definitive endoderm (DE), mesoderm (MD), foregut (lung, thyroid) (AF), hindgut (pancreas), liver (PF), midgut / hindgut (intestine) (M / H), etc.). The names of these pathways are shown in boxes above the activity scores. The pathway activities (also called pathway activity scores) were determined on a log2odds scale. Thus, the values ​​shown are log2odds values. [Figure 1B]FIG. 1B exemplarily shows pathway activity during differentiation of human stem cells (human embryonic stem cells, undifferentiated; hESC undiff) into endoderm by SR-1 induction (SR-1 ind). Mesoderm (MD) is formed by AFBLy differentiation of hESC (AFBLy-diff hESC) (log2odd values ​​after 3 days (3d) are shown). Pathway activity was obtained by Philips pathway analysis of Affymetrix U133Plus2.0 microarray data available in the public GEO dataset GSE52658. FIG. 1B is a schematic diagram showing the successive differentiation steps where specific pathways are activated (after 1 day (1d), 3 days (3d) and 7 days (7d) by SR-1 induction (SR-1 ind)) necessary to obtain each step in the differentiation. Signaling pathway activity is shown as log2 odds value per individual sample for each of the PI3K-FOXO, Hedgehog, TGF-β, Wnt, Notch, and STAT3 pathways. Human embryonic stem cells (hESCs) can be differentiated into various cell types under the control of specific signals. Signaling pathway activity is a readout of such signals, and measuring pathway activity can be a useful tool to evaluate the differentiation / maturation state of cultured cells. hESCs can be differentiated into various cell types (in this example in Figure 1B: foregut (lung, thyroid) (AF), posterior foregut (pancreas, liver) (PF), midgut and hindgut (intestine) (M / H)) by passing through various differentiation steps (anterior primitive streak (APS) and definitive endoderm (DE) in Figure 1B) as indicated by arrows in Figure 1B. For example, the foregut (AF) is formed from the primitive streak via the definitive endoderm. Mesoderm (MD) is formed by AFBLy differentiation of hESCs (AFBLy-diff hESCs) (log2odd values ​​after 3 days (3d) are shown). Signaling pathway activity was measured using Philips signaling pathway analysis in each cell type, resulting in specific pathway signatures. [Figure 2A]FIG. 2A exemplarily shows the results of a differentiation experiment carried out on a non-functionalized surface seeded with WPMY-1 cells. Functionalization was performed with EDC / NHS as disclosed herein. In the TGFbl coating experiment, WPMY1 fibroblasts were seeded and cultured on a functionalized surface in DMEM medium with 10% FBS (SIL-IM-F 0.5%, a silicone substrate containing linoleic acid as described herein and in the previously referenced International Patent Application PCT / EP2018 / 068318), which was coated with TGFbl by incubation with 10 ng of TGFbl (SIL-IM-F 0.5%-TGFbl) according to the described protocol. The non-functionalized surface was also incubated with TGFbl to act as a control. Cells specifically bind to the TGF-β-coated surface, which is believed to depend on the expression of the cellular TGF-β receptor. On uncoated surfaces, cells bind in a specific manner, independent of the expression of a particular receptor, such as the TGF-β receptor. Fewer cells bind to TGF-β-coated surfaces and cell morphology changes, presumably caused by TGF-β-induced activation of the TGF-β signaling pathway (see experiments using TGF-β as an example, further described below). [Figure 2B]FIG. 2B exemplarily shows the results of a differentiation experiment carried out on a functionalized surface coated with TGFbl seeded with WPMY-1 cells. The functionalization was carried out with EDC / NHS as disclosed herein. In the TGFbl coating experiment, WPMY1 fibroblasts were seeded and cultured on a functionalized surface in DMEM medium with 10% FBS (SIL-IM-F 0.5%, a silicone substrate containing linoleic acid as described herein and in the previously referenced International Patent Application PCT / EP2018 / 068318), which was coated with TGFbl by incubation with 10 ng of TGFbl (SIL-IM-F 0.5%-TGFbl) according to the described protocol. The non-functionalized surface was also incubated with TGFbl in the same way and acted as a control. The cells specifically bind to the TGF-β-coated surface, which is believed to be dependent on the expression of the cellular TGF-β receptor. On uncoated surfaces, cells bind in a specific manner, independent of the expression of a particular receptor, such as the TGF-β receptor. Fewer cells bind to TGF-β-coated surfaces and cell morphology changes, presumably caused by TGF-β-induced activation of the TGF-β signaling pathway (see experiments using TGF-β as an example, further described below). [Diagram 3] Figure 3 exemplarily shows an independent cell culture experiment in which the concentration of TGFbl was varied on three different surfaces. A tissue culture treated plastic surface as a control (CT), a non-functionalized silicone surface (NFS) and a functionalized silicone surface (FS) were incubated with different concentrations of TGFbl, 0 ng / ml, 10 ng / ml and 1000 ng / ml (indicated as No, 10 ng and 1 μg, respectively, in Figure 3). On the y-axis, the TGF-β pathway activity score is shown. [Figure 4]FIG. 4 exemplarily shows another independent cell culture experiment in which JAG1 was used as a NOTCH receptor ligand bound to polystyrene (PS), modified silicone (MS) and normal silicone (S) culture plates to specifically activate the Notch pathway in cell culture. The ligand-bound polystyrene culture plates served as positive controls, and the unmodified silicone culture plates served as negative controls. Each experiment involving a ligand (right bar in each of the three pairs) is accompanied by a corresponding experiment without the ligand (corresponding left bar in the three pairs). Pathway activity is shown as a pathway score, according to which the higher the score, the higher the Notch pathway activity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The following embodiments merely illustrate particularly preferred methods and selected aspects related to the methods. The teachings provided herein can be used to construct several test methods, test systems and / or test kits, for example, to generate cell cultures, tissues and / or organs. The following embodiments should not be construed as limiting the scope of the invention.

[0033] The extracellular microenvironment plays an important role in influencing cell behavior, including maintenance of potency, cell attachment, proliferation, metabolism, lineage-specific differentiation, and remodeling. By manipulating the culture conditions in which cells, including stem cells, are cultured, it is possible to influence cell behavior and restrict differentiation pathways, thereby generating cell cultures enriched for lineage-specific precursors, specific tissues, or organs, among others, in vitro. Based on this understanding, one aspect of the present invention relates to substrates, such as scaffolds, for steering cells in a predetermined direction of cellular function.

[0034] An elastomeric material carrying (i.e. binding) one or more stimuli may be denoted as a scaffold or cell culture scaffold, corresponding to the terminology used in the aforementioned international patent application published under number WO2019 / 015988. Such a cell culture scaffold may be produced in a straightforward manner by a simple coupling reaction of said one or more stimuli to reactive groups (e.g. carboxylic acid groups) of an elastomeric material or a precursor elastomeric material, resulting in a cell culture scaffold with a substantially uniform distribution of said one or more stimuli over the surface of the material.

[0035] In one embodiment, the substrate or scaffold is a cell differentiation scaffold for directing cells in a predetermined direction of cell differentiation, the scaffold having a polydimethylsiloxane (PDMS)-, rubber- or silicone-based polymer surface and one or more cell function-inducing stimuli, preferably one or more cell differentiation-inducing stimuli, attached to the polymer surface.

[0036] The term "cell function", also referred to herein as "cell behavior" or "cell behaviour", refers to the functional or morphological response of a cell or cell culture to the applied culture conditions. Exemplary functional responses that may be elicited include, for example, cell division, cell proliferation, differentiation, development, maintenance of pluripotency, etc. Further cell behaviors that may be controlled by the present invention are described herein.

[0037] The term "cell function-inducing stimulus," as disclosed herein, refers to culture conditions provided to induce a predetermined cell function in a cell.

[0038] The one or more cell function inducing stimuli (preferably differentiation inducing stimuli) are preferably selected from the group consisting of cell receptor ligands, extracellular matrix molecules, artificial receptor agonists or antagonists, and specific antigens for T or B cell receptors. Preferably, the one or more cell function inducing stimuli (preferably differentiation inducing stimuli) are ligands that activate signaling through a signaling pathway selected from the group consisting of nuclear receptors such as ER, AR, progesterone receptors; growth factor pathways such as PI3K-FOXO, JAK-STAT3, MAPK-AP1 pathways; immune pathways such as STAT1 / 2 type I interferon (STAT1 / 2-1), STAT1 / 2 type II interferon (STAT1 / 2-2) pathways; developmental pathways such as Hedgehog, Notch, TGF-β, and Wnt signaling pathways; and inflammatory pathways such as NFkB. The ligands may be of natural or non-natural (synthetic) origin. The ligands may be artificial ligands and / or may not occur in nature per se. The ligand may be an artificial and / or synthetic protein ligand, such as an activating antibody or a peptide.

[0039] A further preferred embodiment relates to a substrate as disclosed herein, wherein said one or more cell function-inducing stimuli (preferably differentiation-inducing stimuli) are attached to said polymer surface via carboxyl groups.

[0040] The substrate or scaffold may be of any shape or form. The substrate may be flat or not. It may have reliefs such as corrugated, spiked or other. The substrate may have or consist of a scaffold, where a scaffold is meant to provide a support function that may be of two-dimensional or three-dimensional shape.

[0041] The substrate or scaffold may be part of a culture device. Such devices generally include a culture chamber with a substrate or scaffold. There may be one or more channels connected to the chamber for transporting fluids from or to the chamber. The chamber and substrate may be formed in the device body, and in one embodiment there may be one or more covers removable from the body to access the chamber from the outside. In a preferred embodiment, the substrate is between an upper cover plate and a lower cover plate (e.g., stacked). The substrate may optionally be adapted to adhere to the upper cover plate and / or the lower cover plate. Since there are many such devices, they will not be described in detail here. Examples of how to design culture units and microfluidic devices can be found, for example, in International Patent Application Publication No. WO2019015988. Examples of precursors for the manufacture of substrates and scaffolds can also be found in the above publications, as will be explained in more detail below.

[0042] The scaffold and culture unit (preferably a microfluidic device) are designed to provide a controlled environment for directing cells in a desired direction of cellular function. Due to this compatibility, a third aspect of the invention relates to a method for generating a cell culture, tissue or organ. The method comprises a step (a) of determining first culture conditions controlling the activity of at least one first signaling pathway of the cells to be cultured. According to this, the activity of said at least one first signaling pathway is selected to controllably direct (steer) the cells to be cultured in a first predetermined direction of cellular function. The phrase "controlling the behavior of the cells in a predetermined manner" is understood to describe a step in which the cells to be cultured are controllably directed in a predetermined direction of cellular function. The determination in step (a) does not mean an active experimental step, but may simply mean that information is collected based on in silico prediction, existing knowledge (e.g. published literature and / or databanks) and / or experience of what signaling pathways are active or inactive in the cells (cell type) of interest in order for said cells to acquire a particular cellular behavior (i.e. cellular function). For example, differentiation of a particular stem cell into a particular cell type or tissue is known to involve a cascade of cell signaling pathways (see, e.g., Tsai et al., BMC Cell Biol, 2010, 11:76). Based on this knowledge, it can be determined that these signaling pathways should be sequentially active or inactive to reach the desired progeny.

[0043] The terms "pathway", "signal transduction pathway" and "signaling pathway" are used interchangeably herein. The at least one signaling pathway may mediate a cellular behavior of the cells to be cultured.

[0044] The term "inferred activity" in the context of pathway activity herein refers to a step in which a dimension corresponding to the pathway activity is inferred. The dimension can preferably describe the pathway activity in a semi-quantitative manner, in which the activity can be classified according to, for example, no activity, low activity, high activity, etc., or in a quantitative manner, where the activity is expressed in the form of discrete or continuous values, which correspond to the level of activity or degree of pathway activation. In this way, a semi-quantitative or quantitative comparison in step (d) is possible. The comparison in step (d) allows to determine whether the inferred activity corresponds to the selected activity in order to control the behavior of the cell in a predetermined manner, i.e. to controllably steer the cell in a predetermined direction of cell function. It can thus be determined whether the cultured cells behave in a desired manner, i.e. to steer the cell function in a desired direction, e.g. to differentiate into desired progeny, to maintain pluripotency, etc. If so, the culture conditions can be maintained, otherwise the conditions can be modified to correct any observed deviation from the desired behavior reflected by the pathway activity determined in step (a).

[0045] According to a preferred embodiment, the method further comprises a step (e) of determining second culture conditions based on a comparison of the estimated activity and the selected activity. Preferably, the second culture conditions are determined (i) so as to reduce deviations of the estimated activity from the selected activity. This means that, if the estimated activity of the at least one signaling pathway does not correspond to the expected activity, i.e. the activity determined in step (a), the second culture conditions determined in step (e) may reflect conditions expected to correct any observed deviations from the desired behavior reflected by the pathway activity determined in step (a).

[0046] As another example, the second culture conditions may be determined (ii) to control the activity of at least one second signaling pathway selected to control the cell behavior in a second predetermined manner. For example, the estimated activity corresponds to the expected activity and the second culture conditions determined in step (e) may be substantially identical to the first culture conditions determined in step (a). This may be useful when the cell culture is intended to continue dividing and / or maintain pluripotency. According to another example, the estimated activity corresponds to the expected activity and the second culture conditions are determined in step (e) to control the activity of at least one second signaling pathway selected to control the cell behavior in a second predetermined manner, i.e. to controllably direct the cell in a second predetermined direction of cell function. This may be particularly useful when differentiation of cells into specific progeny involves a cascade of signaling pathways that need to be activated or deactivated in a sequential manner.

[0047] According to a further preferred embodiment, the method further comprises the steps of: (f) culturing the cells in the culture unit under second culture conditions; and (g) repeating steps (c) and (d) as described herein. Preferably, steps (c) and (d) are repeated at intervals. The intervals can be set depending on the cells, the culture conditions and / or the desired cell behavior and can range from a few hours to a few weeks. Suitable intervals are, for example, in the range of 6 hours to 3 weeks, 6 hours to 2 weeks, 6 hours to 10 days, 6 hours to 7 days, preferably 8 hours to 5 days, more preferably 8 hours to 3 days, in particular 8 hours to 2 days, such as once a day.

[0048] Optionally, the method further comprises a step (h) of determining third culture conditions based on a comparison of the inferred activity in step (g) with the selected activity. In this manner, the method allows for feedback control of cell behavior based on pathway activity. The optional step (h) may, for example, reflect conditions that affect the activity of at least one third signaling pathway, e.g., a third signaling pathway involved in a cascade of multiple signaling pathways required for differentiation of a cell into a particular progeny.

[0049] The methods of the present invention allow for the control of cellular behaviors mediated by one or more signaling pathways. Cellular behaviors that can be affected include, but are not limited to, differentiation, maintenance of pluripotency, and cellular functions such as cell division, migration and metabolism, chemotaxis, adhesion, apoptosis, production of oxidative stress, DNA repair, production of secreted proteins, and production of extracellular matrix proteins.

[0050] [Stimulus] According to a preferred embodiment, the first, second and / or third etc. culture conditions are defined by controlled exposure of the cells to one or more cell function inducing stimuli, in which the stimuli control the activity of at least one first and / or second signaling pathway. In other words, the cell culture is exposed to well-defined stimuli that can activate or deactivate the corresponding signaling pathway. Suitable stimuli include naturally occurring substances, but also non-natural substances such as artificial proteins or peptides, drugs that activate or modify pathway activity by binding to surface receptors, such as antibodies such as trastuzumab and antigenic peptides that bind to T-cell or B-cell receptors and initiate cell division via the PI3K pathway. Thus, the one or more cell function inducing stimuli are preferably selected from the group of cell receptor ligands, extracellular matrix molecules, artificial receptor agonists or antagonists, or specific antigens for T- or B-cell receptors.

[0051] According to a preferred embodiment, the one or more cell function inducing stimuli are provided in the form of a scaffold or culture unit, as defined herein. The one or more cell function inducing stimuli can thus be bound to the polymeric surface of the scaffold or culture unit. Binding the one or more stimuli to a surface in the correct three-dimensional arrangement (conformation) in the immediate microenvironment of the cells more closely reflects the in vivo situation, where ligands are often expressed on the outside of the cell membrane of adjacent cells or bound to the extracellular matrix surrounding the cells, than providing them in a solution flowing along the cells attached to a surface. This embodiment can make many ligands very effective.

[0052] The one or more cell function-inducing stimuli are preferably provided in a well-defined spatial and / or temporal manner. To provide the one or more stimuli in a specific spatial distribution, the surface can be patterned with different types of stimuli. For example, two different cell types with different stimuli requirements can be aligned. Furthermore, the concentration of the stimuli can be varied and / or adapted to the specific needs. For example, a low concentration of a particular stimulus can induce cell differentiation, whereas a high concentration of the same stimulus induces cell division. The TGF-β pathway as a typical example determines cell fate in a concentration-dependent manner. In addition to or instead of varying concentrations, a concentration gradient can be envisaged. A mixture of stimuli can be provided when different receptors need to be activated in the cell to obtain a desired effect. An example is the use of growth factor ligands in combination with TGF-β.

[0053] [Surface properties and chemistry] Most of the large-sized tissues and organs with well-defined three-dimensional shapes will require support for their formation from cells. For example, stem cells exist in a specialized microenvironment called the stem cell niche, which provides extracellular stimuli to stem cells to enable their survival and identity. This niche is a key regulator of stem cell behavior, as it guarantees a quiescent, low-metabolic environment to prevent attrition. The microenvironmental properties of the niche are believed to provide a good balance between the ability of stem cells to renew themselves and to differentiate into mature cells so that continuous tissue regeneration occurs. The main part of the cell niche is the ECM (extracellular matrix), which has specific mechanical, biochemical and biophysical properties for tissues and controls the overall cell behavior. The composition of the ECM provides the complete support for the niche through its physical and structural properties. The main function of the artificial support is similar to that of the natural ECM, which supports cell proliferation, differentiation and biosynthesis. For this reason, it is desirable that the surface of the culture unit not only has biochemical (e.g., function-inducing) stimuli, but also has well-defined physical properties, such as well-defined mechanical properties, including surface stiffness / flexibility.

[0054] The physical properties may be provided, at least in part, by the elastomeric material of the substrate or scaffold, which provides a form of stiffness or flexibility. According to IUPAC, an "elastomer" is a polymer or polymeric material that exhibits rubber-like elasticity. Thus, elastomeric materials are also referred to as rubber materials. Elastomeric materials are generally polymeric materials that are viscoelastic (i.e., both viscous and elastic), and generally have a low Young's modulus and a high strain at break compared to other materials. Elastomers are generally polymeric materials, many of which have a polymer backbone held together by intermolecular forces, such as those provided by crosslinks.

[0055] The elastomer to be used in the present invention may be any type of elastomer. For example, the elastomer may be a homopolymer, copolymer, block copolymer, terpolymer, block terpolymer, etc., or a mixture of two or more of these. Particularly suitable elastomers may be selected from polyenes, such as polybutadiene, and / or silicones (polysiloxanes). Suitable polysiloxanes include or consist of polydimethylsiloxane (PDMS). The polysiloxane may comprise a crosslinked siloxane backbone. The crosslinking may be obtained by hydrosilylation reaction known in the art between silylhydride moieties and vinyl moieties of the polysiloxane precursors, using a suitable silylhydride catalyst (e.g. Pt-catalyst).

[0056] In one embodiment, the elastomeric material comprises or consists of a polysiloxane and / or polydiene material. Preferably, the polysiloxane comprises or consists of polydimethylsiloxane (PDMS). A preferred polydiene material is polybutadiene.

[0057] Many elastomers are known in the art. However, the elastomeric materials of the present invention are modified in that they have stimuli attached to them (i.e., the elastomeric materials bear stimuli). At least a portion of such attached stimuli must be available at the elastomeric surface. In this way, they are available to provide the desired stimuli to cells during cell culture. Attachment in this respect can mean any type of chemical or physical attachment, but preferably includes or consists of a covalent bond via a linking chemical group. Examples of such modifications and attachments are described below.

[0058] Many of the stimuli that are coupled to the elastomers are organic molecules and also proteins. As is known, many proteins have carboxylic acid or amine-containing amino acids on their periphery that can be used for chemical conjugation or labeling. In principle, any type or method of conjugation that maintains the stimuli function of such organic molecules or proteins when coupled to the elastomeric material can be used. In the field of chemistry known as bioconjugation reactions, many different coupling reactions are known that lead to various linking groups. See, for example, "Surface modification Chemistries of Materials used in diagnostic platforms with biomolecules" by MD Sonawane and SB Nimse, published in Journal of Chemistry, vol. 16, page 1 e.v., and ThermoScientific Crosslinking Handbook; "easy molecular bonding crosslinking technology, Reactivity chemistries, applications and structure references, 2012", available for download at the internet page http: / / tools.thermofisher.com / content / sfs / brochures / 1602163-Crosslinking-Reagents-Handbook.pdf. The latter reference in particular lists a variety of chemistries that can be used for coupling in the present invention. In particular, the paragraph on immobilization of proteins to solid supports provides guidance on how to couple proteins to solid surfaces with carboxy- and amine-modified elastomeric polymers. An example of a suitable coupling chemistry would be based on N-hydroxysuccinimide (NHS) esters as activated carboxylate functionalities for coupling to, for example, primary amines.Often a carbodiimide such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or N,N'-dicyclohexylcarbodiimide (DCC) is used to activate the carboxylic acid group for reaction with NHS at an intermediate step.

[0059] To allow such binding to occur, precursor elastomeric materials can be used that are functionalized with reactive groups for binding of the stimuli, for example using chemistry as described above. Examples of reactive groups can be selected from carboxylic acids or conjugate bases, carboxylates, and amine groups, but others can be used. Thus, for example, carboxylic acid groups of the elastomeric material precursor can be used to bind to amine groups of the stimuli, or vice versa. Functionalization can be achieved in different ways. A first group of elastomeric precursor materials may have functional groups available for further reaction after formation (polymerization) (e.g. polybutadiene), while other groups require explicit functionalization (e.g. many polysiloxanes such as PDMS). Functionalization can be achieved in different ways. For example, precursor elastomeric materials such as PDMS can be surface treated (after their polymerization) using different types of treatments, for example oxidation by plasma. These are techniques well known in the art. In a further method, suitable chemical groups are incorporated into the precursor elastomeric materials during their formation by polymerization. Such methods are, for example, described herein and have been used in the manufacture of the experimental samples described herein.

[0060] In one embodiment, the elastomeric material comprises or consists of a polysiloxane and / or polydiene material, where the one or more stimuli are attached to the surface via carboxyl groups. Advantageously, attaching the one or more stimuli to carboxyl groups protruding from a surface material such as PDMS or Lucas rubber mimics the in vivo situation for, for example, cell receptor ligands of members of the TGF-β family, ligands of the Hedgehog and Notch pathways, and ligands of integrin-induced cell signaling. As a further advantage, changes in substrate stiffness can be easily obtained in both rubber and PDMS.

[0061] According to a preferred embodiment, the surface comprises or consists of a material for binding said one or more stimuli, such as cell receptor ligands, in which case the material corresponds to the material for binding cell culture proteins as described in International Patent Application PCT / EP2018 / 068318 entitled "Cell culturing materials", published as PCT patent application WO2019 / 015988, which claims priority to EP application number 17181708.5, filed July 18, 2017, and has a filing date of July 6, 2018 (the disclosure of which is incorporated herein by reference in its entirety). See in particular the surface materials and the surface chemistries which make the surface materials suitable for binding cell culture proteins. The materials disclosed therein are capable of binding one or more stimuli as described herein, instead of the cell culture proteins as described in said International Patent Application.

[0062] According to a further preferred embodiment, the scaffold disclosed herein is a scaffold obtained by functionalizing the polymer surface prior to binding one or more functionalizing stimuli. The functionalization is preferably carried out using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) followed by reaction with N-hydroxysuccinimide (NHS), as disclosed herein or in PCT patent application WO2020 / 015888.

[0063] EDC / NHS are initiators (catalysts) that are washed away after binding the functional protein. These initiators are standard. They are not detectable after functionalization.

[0064] For purposes of this description and experiment, the material includes, inter alia, bulk modified elastomers having multiple fatty acid moieties covalently bonded to the elastomer bulk, the carboxylic acid (carboxylic acid) groups of said moieties being available for binding to said one or more stimuli. Such materials can advantageously be manufactured in several steps. In some embodiments, a single step is sufficient, for example, when the manufacturing is performed by combining the elastomer and the fatty acid in a coating process (e.g., spin coating, dip coating, spray coating, dispensing) or injection molding process, in which case crosslinking of the elastomer with the fatty acid carbon-carbon double bonds can be achieved without significant epoxidization of the fatty acid carbon-carbon double bonds due to the limited exposure to ambient oxygen in the coating or injection molding process. This provides materials in which the elastomer is bulk modified with fatty acid moieties, the carboxylic acid groups of the fatty acid being available for binding to a biocompatible material, such as one or more stimuli described herein.

[0065] Furthermore, by controlling the curing and / or properties of the mold in which the material is formed, such carboxylic acid groups can be present primarily on the outer surface of the material, resulting in a substantially uniform distribution of carboxylic acid groups on such outer surface, which makes the material particularly suitable for use as a membrane material for fluidic devices, since each cross-section of the material exhibits the same surface properties, in contrast to membrane materials to which anchors for biocompatible materials must be grafted or otherwise formed as per known techniques. In addition, since only a small portion of the elastomeric carbon-carbon double bonds are typically consumed in such cross-linking reactions, the material retains the elastomeric properties of the elastomer, which enhances the suitability of the material for use in fluidic devices and makes it easier to slice or cut the material for investigation or other purposes.

[0066] Preferably, each of the fatty acid moieties is covalently attached to the elastomer bulk via a cross-linking reaction between a vinyl or hydride functional group and an unsaturated carbon-carbon bond of the unsaturated fatty acid to ensure that the number of carboxylic acid groups available for attachment to a biocompatible material can be optimized.

[0067] The elastomer may be any suitable elastomer. For example, the elastomer may be a homopolymer, copolymer, block copolymer, terpolymer, block terpolymer, etc. Particularly suitable elastomers may be selected from polyenes such as polybutadiene or silicones (polysiloxanes). Such silicones may have a PDMS backbone that includes vinyl moieties to promote crosslinking via Pt-catalyzed addition reactions, for example with (polymethyl)hydrogensiloxanes. As another example, silicone backbones such as (polymethyl)hydrogensiloxane backbones may be crosslinked with rubber-like polymers such as polybutadiene and polyisoprene, with unsaturated fatty acids being incorporated into such crosslinked products.

[0068] As for the unsaturated fatty acid, any suitable unsaturated fatty acid can be used for crosslinking with the elastomer.For example, the unsaturated fatty acid can be selected from myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoellagic acid, α-linolenic acid, arachidonic acid, eicosperethane acid, erucic acid and docosahexaenoic acid.Particularly, linoleic acid can be mentioned.

[0069] The elastomer and unsaturated fatty acid may be mixed in any suitable ratio in a composition to form the bulk modified elastomer. Preferably, the unsaturated fatty acid is present in such a composition in the range of 0.05-35% by weight of the elastomer, such that upon bulk modification of the elastomer, the bulk modified elastomer retains its flexibility due to the remaining presence of carbon-carbon double bonds in the bulk of the elastomer.

[0070] It will be apparent that other methods of obtaining stimuli-coupled elastomeric materials may also be usefully employed in achieving the cell functionalization described above.

[0071] If patterning of stimulation as described above is desired, a fluidic device with two or more membrane bodies can be used, in which case each membrane body can be advantageously manufactured as described above. The membrane bodies can be stacked so that cells can be cultured in front of each other. In this way, for example, liver tissue and pancreatic tissue can be grown in front of each other. The consumption of glucose in the nutrition of both organs and the production of insulin by the pancreas can be studied in the co-operating organ on a chip system.

[0072] [Microfluidic Devices] Control of cell behavior, such as stem cell culture and differentiation, requires precise control of multiple stimuli in the cell culture microenvironment that modulate intracellular signaling and ultimately cell phenotype. Microfluidic devices that allow for precise control of multiple stimuli simultaneously over space and time provide an ideal and well-defined platform for culturing cells, such as stem cells, in a controlled manner.

[0073] According to a preferred embodiment, the cell culture scaffold is part of a fluidic device module that includes at least one channel extending over a membrane body carrying the cell culture scaffold. That is, the culture unit is a microfluidic device. Such a fluidic device module may have a first main surface that includes a first concave structure that defines a first flow path. Depending on the application, a second main surface opposite the first main surface and having a second concave structure that defines a second flow path may be envisaged as the membrane body separating the first and second flow paths. This configuration is particularly useful when the cell culture has to be used later in the same device to monitor the response of cells to drugs, as described in the aforementioned international patent application. In a preferred embodiment, the fluidic device module is a monolithic fluidic device module, which has the advantage that the device module can be manufactured by injection molding in a few processing steps (e.g. a single processing step).

[0074] The membrane may have a number of holes or channels extending therethrough, preferably with cell-sized dimensions to retain the cells to be cultured.

[0075] In one embodiment, the first flow channel and the second flow channel are accessible through corresponding partitions, allowing separate flow channels to be individually accessed without risk of cross-contamination.

[0076] The fluidic device module may further comprise a pair of cover plates (e.g. transparent glass sheets), where one cover sheet preferably has two inlets and two outlets to fluidically seal the fluidic device module. The cover plates may be arranged such that one of the cover plates covers the first main surface, thereby sealing the first flow path, while the other cover plate covers the second main surface, thereby sealing the second flow path. Such a fluidic device module can be manufactured in a simple manner, since it can be formed in a small number of processing steps as described above, and is robust against leakage due to the flexible nature of the fluidic device module. When the membrane body is formed of an elastomeric material, in particular a soft and flexible elastomeric material, the cover plates advantageously form a leak-free fluidic device module together with the membrane body clamped between the cover plates.

[0077] In a preferred embodiment, the membrane can be glued to the top and / or bottom cover plate. As a result, after clamping the membrane and cover plate together, the adhesive properties of the membrane keep the parts together. This allows the fluidic device module to be handled under / on a microscope, such as a confocal microscope, after removal from a fluidic system, such as Micronit or Fluigent, without the risk of the cover plate (formed from a particularly transparent material to facilitate microscopic examination) peeling off from the membrane.

[0078] It is also envisaged that the membrane may allow for easy removal of the upper cover plate, whilst being attached to a lower cover plate such that the cell culture, organ, tissue or biopsy on the membrane may be curved towards the lower cover plate, which remains in contact with the underside of the membrane, allowing it to be accurately positioned at the focus of a confocal microscope.

[0079] To change the culture conditions from a first culture condition to a second culture condition etc. at different times, the cells grown on a first surface and / or membrane body can be detached and replaced with a second surface and / or membrane body carrying one or more second stimuli. Advantageously, surfaces and / or membrane bodies pre-coated with a number of different stimuli can be prepared and provided in kit format for this purpose.

[0080] For detailed descriptions of suitable methods of manufacture, other suitable unsaturated fatty acids and elastomers, structural modifications of the membrane and / or specifics regarding the construction of the fluidic device modules, please refer to the international patent applications cited above. [Cell type]

[0081] The cell is not particularly limited and can be any cell or cell line aimed at obtaining a particular cellular behavior. According to a particular embodiment, the cell is selected from the group consisting of mouse stem cells, human stem cells, pluripotent stem cells, mesenchymal stem cells, embryonic stem cells, adult stem cells and tumor cells.

[0082] Stem cells have great potential in many clinically relevant applications including tissue engineering, organ regeneration, cell-based therapy, disease modeling, drug response testing, drug screening and development, and various health care applications. The major step in most applications involving stem cells is to direct the differentiation of the cells into the desired progeny. There are, of course, two types of stem cells, the first being embryonic stem cells (ESCs) and the second being adult stem cells. ESCs are isolated from the inner cell mass of the blastocyst and have the pluripotency to differentiate into virtually all cell lineages. Adult stem cells can be found in a variety of tissues and can differentiate into a limited number of cell types. In addition, there are reprogrammed human induced pluripotent stem cells (hiPSCs) that can be obtained directly from adult cells by introducing specific genes. These represent a single source of cells that can be used to replace cells lost due to injury or disease, as they can multiply indefinitely and give rise to all other cell types in the body. In certain embodiments, the cells employed in the present method do not require the destruction of an embryo. In such certain embodiments, embryonic stem cells are excluded from the scope of the present invention.

[0083] The tumor cells may be used to generate cancer model systems for the purposes of diagnosing or subtyping cancer and / or for testing the response of cancer cells to drugs.

[0084] [Pathway analysis] According to a further preferred embodiment, the at least one signaling pathway is selected from the following group: - Nuclear receptors such as ER, AR, and progesterone receptor; - Growth factor pathways such as the PI3K-FOXO, JAK-STAT3, and MAPK-AP1 pathways; - immune pathways such as the STAT1 / 2 type I interferon (STAT1 / 2-1) and STAT1 / 2 type II interferon (STAT1 / 2-2) pathways; - Developmental pathways such as Hedgehog, Notch, TGF-β, and Wnt signaling pathways; and - Inflammatory pathway NFkB.

[0085] According to a further preferred embodiment, the activity of at least one first and / or second etc. signaling pathway in a cell sample can be inferred by a method comprising the steps of: - receiving expression levels of three or more target genes of said at least one signaling pathway in at least one cell contained in said cell sample; - determining an activity level of a transcription factor (TF) element in said at least one cell, said TF element controlling transcription of said three or more target genes, said determining being based on evaluating a calibrated mathematical pathway model relating expression levels of said three or more target genes to activity levels of said at least one signaling pathway; and - inferring an activity of said at least one signaling pathway in said at least one cell based on the determined activity level of said TF element; Here, the calibrated mathematical path model is preferably a centroid, linear or Bayesian network model based on conditional probability.

[0086] The term "activity of at least one signaling pathway" may refer to the activity of a signaling pathway-associated transcription factor (TF) element in the sample in driving the target gene into expression, i.e., the rate at which the target gene is transcribed, e.g., in terms of high activity (i.e., high rate) or low activity (i.e., low rate), or a corresponding value, level, score, dimension, etc., associated with or indicative of such activity (e.g., rate). Thus, for the purposes of the present invention, the term "activity" as used herein is also intended to refer to said activity level. Both activity and activity level can be determined by pathway analysis as described herein. As mentioned above, activity can be the same as activity level. Such level can be directly used as input for step (d).

[0087] Pathway activity can be determined, for example, in samples isolated from cell cultures by pathway analysis as described herein.

[0088] Pathway analysis allows the quantitative measurement of signaling pathway activity in cells present in a tissue / cell sample based on inferring signaling pathway activity from the measurement of mRNA levels of well-validated direct target genes of transcription factors associated with the corresponding signaling pathway (see e.g. W Verhaegh et al. “Selection of personalized patient therapy through the use of knowledge-based computational models that identify tumor-driving signal transduction pathways” Cancer research 2014; 74(11):2936-45; W Verhaegh, A van de Stolpe “Knowledge-based computational models” Oncotarget 2014; 5(14):5196).

[0089] According to preferred embodiments of the various aspects of the invention and various embodiments thereof, determining the activity of one or more pathways, combinations of pathway activities, and applications thereof, as disclosed herein, may be carried out, for example, as described in the following documents, each of which is incorporated herein in its entirety, for purposes of determining the activity of each signaling pathway: Namely, published international patent applications WO2013011479 (titled "ASSESSMENT OF CELLULAR SIGNALING PATHWAY ACTIVITY USING PROBABILISTIC MODELING OF TARGET GENE EXPRESSION"); WO2014102668 (titled "ASSESSMENT OF CELLULAR SIGNALING PATHWAY ACTIVITY USING LINEAR COMBINATION(S) OF TARGET GENE EXPRESSIONS"); WO2015101635 (titled "ASSESSMENT OF THE PI3K CELLULAR SIGNALING PATHWAY ACTIVITY USING MATHEMATICAL MODELLING OF TARGET GENE EXPRESSION"); WO2016062891 (titled "ASSESSMENT OF TGF-β CELLULAR SIGNALING PATHWAY ACTIVITY USING MATHEMATICAL MODELLING OF TARGET GENE EXPRESSION"); WO2017029215(Name “ASSESSMENT OF NFKB CELLULAR SIGNALING PATHWAY ACTIVITY USING MATHEMATICAL MODELLING OF TARGET GENE EXPRESSION”);W02014174003(Name “MEDICAL PROGNOSIS AND PREDICTION OF TREATMENT RESPONSE USING MULTIPLE CELLULAR SIGNALLING PATHWAY ACTIVITIES”);No. WO2016062892 (filed Nov 25, 2016; entitled "MEDICAL PROGNOSIS AND PREDICTION OF TREATMENT RESPONSE USING MULTIPLE CELLULAR SIGNALING PATHWAY ACTIVITIES"); No. WO2016062893 (filed Oct 25, 2016; entitled "Method to distinguish tumor suppressive FOXO activity from oxidative stress"); No. EP17194288.1 (filed Oct 2, 2017; entitled "Assessment of Notch cellular signaling pathway activity using mathematical modelling of target gene expression"); No. EP17194291.5 (filed Oct 2, 2017; entitled "Assessment of Notch cellular signaling pathway activity using mathematical modelling of target gene expression"); 2017; Name “Assessment of JAK-STAT1 / 2 cellular signaling pathway activity using mathematical modeling of target gene expression”); EP17194293.1 (Application filed Oct 2, 2017; Name “Assessment of JAK-STAT3 cellular signaling pathway activity using mathematical modeling of target gene expression”);and EP17209053.2 (filed Dec 20, 2017, entitled "Assessment of MAPK-APl cellular signaling pathway activity using mathematical modelling of target gene expression"). The models have been biologically validated in several cell types for the ER, AR, PI3K-FOXO, HH, Notch, TGF-β, Wnt, NFkB, JAK-STAT1 / 2, JAK-STAT3 and MAPK-AP1 pathways. It is noted that the mathematical models employed in the as yet unpublished patent applications, as well as the calibration and use of these models in these applications, generally correspond to the models, calibration and use disclosed in the already published patent applications.;

[0090] To facilitate the quick identification of the references, the above references are assigned to each signaling pathway of interest herein, and exemplary corresponding target genes suitable for determining the activity of said signaling pathway are indicated. In this regard, special reference is also made to the sequence list of target genes provided in the above references, namely, Wnt: KIAA1199, AXIN2, RNF43, TBX3, TDGF1, SOX9, ASCL2, IL8, SP5, ZNRF3, KLF6, CCND1, DEFA6 and FZD7 (WO2013 / 011479, WO2014 / 102668, WO2014 / 174003); ADRA2C, ASCL2, AXIN2, BMP7, CCND1, CD44, COL18A1, DEFA6, DKK1, EPHB2, EPHB3, FAT1, FZD7, GLUL, HNFIA, CXCL8 (previously known as IL8), CEMIP (KIAA1 199), KLF6, LECT2, LEF1, LGR5, MYC, NKD1, OAT, PPARG, REGIB, RNF43, SLC1A2, SOX9, SP5, TBX3, TCF7L2, TDGF1 and ZNRF3 (WO2016 / 062892, WO 2016 / 062893);HH: GLI1, PTCH1, PTCH2, IGFBP6, SPP1, CCND2, FST, FOXL1, CFLAR, TSC22D1, RAB34, S100A9, S100A7, MYCN, FOXM1, GLI3, TCEA2, FYN and CTSL1 (WO2013 / 011479, WO2014 / 102668, WO2014 / 174003);GLI1, PTCH1, PTCH2, HHIP, SPP1 ,TSC22D1,CCND2,HI9,IGFBP6,TOM1,JUP,FOXA2,MYCN,NKX2-2,NKX2-8,RAB34,MIF,GLI3,FST,BCL2,CTSL1,TCEA2,MY LK, FYN, PITRM1, CFLAR, IL1R2, S100A7, S100A9, CCND1, JAG2, FOXM1, FOXF1 and FOXL1 (WO2016 / 062892, WO2016 / 062893);AR:KLK2,PMEPA1,TMPRSS2,NKX3 1,ABCC4,KLK3,FKBP5,ELL2,UGT2B15,DHCR24,PPAP2A,NDRG1,LRIG1,CREB3L4,LCP1,GUCY1 A3,AR and EAF2(WO2013 / 011479,WO2014 / 102668);KLK2,PMEPA1,TMPRSS2,NKX3 1,ABCC4,KLK3,FKBP5,ELL2,UGT2B 15,DHCR24,PPAP2A,NDRGl,LRIG1,CREB3L4,LCP1,GUCY1 A3,AR AND EAF2(WO2014 / 174003);ER:CDH26,SGK3,PGR,GREBl,CA12,XBP1,CELSR2,WISP2,DSCAM,ERBB2,CTSD,TFF1 AND NRIPl(WO2013 / 011479,WO2014). / 102668);GREBl,PGR,XBP1,CA12,SOD1,CTSD,IGFBP4,TFF1,SGK3,NRIPl,CELSR2,WISP2 and APIBI(WO2014 / 174003);APIBI,ATP5J,COL18A1,COX7A2L ,CTSD,DSCAM,EBAG9,ESR1,HSPB1,KRT19,NDUFV3,NRIPl,PGR,PISD,PRDM15,PTMA,RARA,SOD1,TFF1,TRIM25,XBP1,GREBl,IGFBP4,MYC,SGK3,WISP2 ,ERBB2,CA12,CDH26AND CELSR2(WO2016 / 062892,WO2016 / 062893);PI3K-FOXO:AGRP,BCL2L11,BCL6,BNIP3,BTG1,CAT,CAV1,CCND1,CCND2,CCNG2,CDK 1A,CDK IB,ESR1,FASLG,FBX032,GADD45A,INSR,MXI1,NOS3,PCK1,POMC,PPARGCIA,PRDX3,RBL2,SOD2 and TNFSF10(WO2015 / 101635);ATP8A1, BCL2L11, BNIP3, BTG1, ClOorflO, CAT, CBLB, CCND1, CCND2, CDKN1B, DDB1, DYRK2, ERBB3, EREG, ESR1, EXT1, FASLG, FGFR2, GADD45A, IGF1R, IGFBP1, IGFBP3, INSR, LGMN, MXI1, PPM1D, SEMA3C, SEPP1, SESN1, SLC5A3, SMAD4, SOD2, TLE4 and TNFSF10 (WO2016 / 062892, WO2016 / 062893); SOD2, BNIP3, MXI1, PCK1, PPARGC1A and CAT (the above EP16200697.7); TGF-β: ANGPTL4, CDC42EP3, CDKN1A, CDKN2B, CTGF, GADD45A, GADD45B, HMGA2, ID1, IL11, SERPINE1, INPP5D, JUNB, MMP2, MMP9, NKX2-5, OVOL1, PDGFB, PTHLH, SGK1, SKIL, SMAD4, SMAD5, SMAD6, SMAD7, SNAI1, SNAI2, TEMPI and VEGFA (WO2016 / 062891, WO2016 / 062893); NFkB: BCL2L1, BIRC3, CCL2, CCL3, CCL4, CCL5, CCL20, CCL22, CX3CL1, CXCL1, CXCL2, CXCL3, ICAM1, IL1B, IL6, IL8, IRF1, MMP9, NFKB2, NFKBIA, NFKBIE, PTGS2, SELE, STAT5A, TNF, TNFAIP2, TNIP1, TRAF1 and VCAM1 (WO2017 / 029215); Notch: CD28, CD44, DLGAP5, DTX1, EPHB3, FABP7, GFAP, GIMAP5, HES1, HES4, HES5, HES7, HEY1, HEY2, HEYL, KLF5, MYC, NFKB2, NOX1, NRARP, PBX1, PIN1, PLXND1, PTCR1A, SOX9 and TNC (the above EP17194288.1);JAK-STAT1 / 2: BID, GNAZ, IRF1, IRF7, IRF8, IRF9, LGALS1, NCF4, NFAM1, OAS1, PDCD1, RAB36, RBX1, RFPL3, SAMM50, SMARCB1, SSTR3, ST13, STAT1, TRMT1, UFDIL, USP18 and ZNRF3, preferably from the group consisting of IRF1, IRF7, IRF8, IRF9, OAS1, PDCD1, ST13, STAT1 and USP18 (see above EP17194291.5); JAK-STAT3: AKT1, BCL2, BCL2L1, BIRC5, CCND1, CD274, CDKN1A, CRP, FGF2, FOS, FSCN1, FSCN2, FSCN3, HIF1A, HSP9 0AA1, HSP90AB1, HSP90B1, HSPA1A, HSPA1B, ICAM1, IFNG, IL10, JunB, MCL1, MMP1, MMP3, MMP9, MUC1, MYC, NOS2, POU2F1, PTGS2, SAA1, STAT1, TIMP1, TNFRSF1B, TWIST1, VIM and ZEB1 (see above EP17194293.1); MAPK-AP1: BCL2L11, CCND1, DDIT3, DNMT1, EGFR, ENPP2, EZR, FASLG, FIGF, GLRX, IL2, IVL, LOR, MMP1, MMP3, MMP9, SERPINE1, PLAU, PLAUR, PTGS2, SNCG, TIMP1, TP53 and VIM (see above EP17209053.2).

[0091] Common to the pathway analysis methods for determining the activity of different signaling pathways disclosed herein is the concept preferably applied herein for the purposes of the present invention, where the activity of a signaling pathway in a cell present in a sample can be determined by: receiving the expression level of one or more, preferably three or more, target genes of said signaling pathway; determining the activity level of a signaling pathway associated transcription factor (TF) element in said sample, where said TF element controls the transcription of said three or more target genes, said determining being based on evaluation of a calibrated mathematical pathway model relating the expression levels of said three or more target genes to the activity level of said signaling pathway; and, optionally, estimating the activity of the signaling pathway in said cell based on said determined activity level of a signaling pathway associated TF element in said sample.

[0092] The term "transcription factor element" (TF element) as used herein preferably refers to an intermediate or precursor protein or protein complex of an active transcription factor, or an active transcription factor protein or protein complex that controls a specific target gene expression. For example, the protein complex may contain at least one intracellular domain of each signaling pathway protein, together with one or more cofactors, thereby controlling the transcription of the target gene. Preferably, the term also refers to either a protein or protein complex transcription factor that is activated by the cleavage of one of the signaling pathway proteins that results in the intracellular domain.

[0093] As indicated above, the term "activity level" of a TF element as used herein refers to the level of activity of the TF element with respect to the transcription of its target gene.

[0094] As used herein, the term "target gene" refers to a gene whose transcription is directly or indirectly controlled by a corresponding transcription factor element. A "target gene" can be a "direct target gene" and / or an "indirect target gene" (as described herein).

[0095] Furthermore, specific reference is made to the definitions of each pathway, each transcription factor element, (set of) target genes, calibration methods (using as calibrated mathematical pathway models a model calibrated using a ground truth data set comprising the following samples: samples in which the transcription of some, e.g. three or more target genes of each cell signaling pathway is induced by each TF element; and samples in which the transcription of some, e.g. three or more target genes is not induced by each TF element) of each cell signaling pathway.

[0096] The calibrated mathematical pathway model may be a probabilistic model, preferably a Bayesian network model, based on conditional probabilities relating the activity levels of the signaling pathway-associated TF elements and the expression levels of the three or more target genes, or the calibrated mathematical pathway model may be based on one or more linear combinations of the expression levels of the three or more target genes. For the purposes of the present invention, the calibrated mathematical pathway model is preferably a centroid or linear model, or a Bayesian network model based on conditional probabilities.

[0097] In particular, said determining of expression levels and optionally inferring activity of the signaling pathway in said cell sample can be performed, for example, inter alia, by: (i) evaluating a portion of a calibrated probabilistic pathway model (preferably a Bayesian network) representing the cell signaling pathway with respect to a set of inputs comprising expression levels of three or more target genes of the cell signaling pathway measured in the cell sample; (ii) estimating activity levels of signaling pathway-associated transcription factor (TF) elements in the cell sample, where said signaling pathway-associated TF elements control transcription of said three or more target genes of the cell signaling pathway, said estimation being based on a conditional probability relating activity levels of signaling pathway-associated TF elements and expression levels of said three or more target genes of the cell signaling pathway measured in the cell sample; and, optionally, (iii) inferring activity of the cell signaling pathway based on the estimated activity levels of signaling pathway-associated TF elements in the cell sample. This is described in detail in published international patent application WO2013 / 011479 A2 ("Assessment of cellular signaling pathway activity using probabilistic modeling of target gene expression"), the contents of which are incorporated herein in their entirety.

[0098] In an exemplary alternative, the determination of the expression level and optionally the inference of the activity of the cell signaling pathway may be performed by, inter alia: (i) determining the activity level of a signaling pathway-associated transcription factor (TF) element in a cell, where the signaling pathway-associated TF element controls the transcription of said three or more target genes of the cell signaling pathway, the determination being based on evaluating a calibrated mathematical pathway model relating the expression levels of said three or more target genes of the cell signaling pathway to the activity level of the signaling pathway-associated TF element, where the mathematical pathway model is based on one or more linear combinations of the expression levels of said three or more target genes; and, optionally, (ii) inferring the activity of the cell signaling pathway in the cells of the cell culture based on the determined activity level of the signaling pathway-associated TF element in the cell sample. This is described in detail in published international patent application WO2014 / 102668 A2 ("Assessment of cellular signaling pathway activity using linear combination(s) of target gene expressions").

[0099] Further details on predicting cell signaling pathway activity using mathematical modeling of target gene expression can be found in the article by Verhaegh W. et al., “Selection of personalized patient therapy through the use of knowledge-based computational models that identify tumor-driving signal transduction pathways,” Cancer Research, Vol. 74, No. 11, 2014, pp. 2936-2945.

[0100] In one embodiment, the measurement of the signaling pathway is performed using qPCR, multiple qPCR, multiplexed qPCR, ddPCR, RNAseq, RNA expression arrays, or mass spectrometry. For example, gene expression microarray data, e.g., Affymetrix microarrays, or RNA sequencing methods such as Illumina sequencers can be used.

[0101] [Storage media, computer programs and systems] According to a fourth aspect of the invention, a non-transitory storage medium stores instructions executable by a digital processing device to perform the method according to the third aspect of the invention and its various embodiments. The non-transitory storage medium may be a computer readable storage medium such as a hard drive or other magnetic storage medium, an optical disk or other optical storage medium, a random access memory (RAM), a read only memory (ROM), a flash memory or other electronic storage medium, a network server, etc. The digital processing device may be a handheld device (e.g., a personal digital assistant or smartphone), a notebook computer, a desktop computer, a tablet computer or device, a remote network server, etc.

[0102] According to a fifth aspect of the invention, a computer program comprises program code means for causing a digital processing apparatus to perform the method according to the third aspect of the invention and its various embodiments when the computer program is executed on the digital processing apparatus, which may be a handheld device (e.g. a personal digital assistant or a smartphone), a notebook computer, a desktop computer, a tablet computer or device, a remote network server, etc.

[0103] According to a sixth aspect of the invention, a system for carrying out the method of the invention comprises: (a) a culture unit or scaffold, preferably as defined herein; (b) a sensor unit for inferring an activity of at least one first signaling pathway; (c) a controller configured to compare the estimated activity of the at least one first signaling pathway with a predetermined activity selected to control the behavior of the cell in a first predetermined manner, i.e., to controllably steer the cell in a predetermined direction of cellular function; and, optionally, (d) a non-transitory storage medium of the present invention or a computer program of the present invention; has.

[0104] According to a preferred embodiment, the controller is further configured to determine culture conditions based on a comparison of the estimated activity and the selected activity, the culture conditions being optionally determined to (i) reduce deviation of the estimated activity from the selected activity or (ii) control activity of at least one second signaling pathway.

[0105] The culture unit is preferably configured as disclosed herein and as described herein in the context of various aspects of the present invention. The sensor unit is adapted to infer the activity of the at least one first signal transduction pathway. For this purpose, the sensor unit may comprise one or more elements or means for measuring the expression level of target genes, such as DNA array chips, oligonucleotide array chips, protein array chips, antibodies, multiple probes (e.g., labeled probes), a set of RNA reverse transcriptase sequencing components, and / or RNA or DNA containing cDNA or amplification primers. Preferably, the sensor unit is adapted to perform one of qPCR, multiple qPCR, multiplexed qPCR, ddPCR, RNAseq, RNA expression arrays, and mass spectrometry.

[0106] In one embodiment, the sensor unit uses a set of labeled probes directed to a portion of the mRNA or cDNA sequence of the target gene as described herein. In one embodiment, the sensor unit uses a set of primers and probes directed to a portion of the mRNA or cDNA sequence of the target gene. In one embodiment, the labeled probes are contained in a standardized 96-well plate. In one embodiment, the sensor unit further uses primers or probes directed to a set of reference genes. Such reference genes can be, for example, constitutively expressed genes useful for normalizing or standardizing the expression levels of the target gene expression levels described herein.

[0107] The sensor unit may further include a processor for calculating a result corresponding to the estimated activity of the at least one first signaling pathway.

[0108] [use] Various aspects of the present invention include: - determining, predicting or controlling the acquisition of specific cellular behaviors in cell cultures; - determining, predicting or controlling that a cell culture has differentiated into or towards a desired progeny; - determining, predicting or controlling the acquisition of a specific function by a cell culture; - determining, predicting or controlling cell culture responses to specific stimuli; - Generate cell cultures, tissues or organs in a controlled and robust manner; - using the cell cultures, tissues or organs produced by the methods of the invention to predict, monitor or determine response to treatment; - using the cell cultures, tissues or organs produced by the methods of the invention to predict, monitor or determine the efficacy of a treatment; - Creating tumor model systems; - diagnosing or subtyping diseases, in particular oncological diseases; - Generate cells suitable for regenerative medicine purposes; - generating a set of cells that are quantitatively very well defined with respect to cell state (e.g. differentiation state) to enable reproducible subsequent experimental use, e.g. in drug development or regenerative medicine; This is particularly useful for

[0109] [Example of differentiation of stem cells into intestinal stem cells] Stem cells (eg, iPS or HES cells) can be differentiated into intestinal stem cells in standard culture according to the protocol described by Loh et al. (supra).

[0110] Supplying cell cultures with ligands (including those that cannot be added to the medium, such as Notch ligands) in a controlled manner in accordance with embodiments of the invention to activate each signaling pathway predicted to be active during the various differentiation stages (see FIG. 1, Pathway analysis of GEO dataset, differentiation into hindgut; A. Pathway activity measured at each differentiation step; B. Overview showing successive differentiation steps; Signaling pathway activity shown as log2 odds values ​​for each individual sample), improves the results of differentiation experiments and makes such experiments more reproducible across laboratories and time, making the method particularly useful for experimental standardization purposes.

[0111] An exemplary experiment according to the invention is performed as follows: The experiment is designed to progress cells through several differentiation steps to obtain first a preprimitive streak cell, followed by a definitive endoderm and then a midgut / hindgut cell phenotype.

[0112] Stem cells are cultured feeder-free at the required density in defined serum-free CDM2 basal medium on tissue culture plastic. Then, for differentiation experiments, they are passaged and seeded on the surface material described herein functionalized with a 50 / 50 mixture (10 micrograms / ml) of the appropriate Wnt ligand (e.g., Wnt3A), activin, in the presence of an mTOR inhibitor, e.g., LDN-193189 / DM3189, 50 nM) in CDM2 medium for 24 hours to obtain preprimitive streak cells. Then, the cells are detached from the surface and a pathway analysis is performed on the cell samples for all the mentioned signaling pathways to ensure that the cells have reached the required differentiation stage to the required quantitative extent. If this pathway analysis gives a satisfactory result, this allows a positive decision to continue the experiment. Then, the cells are seeded on the next substrate functionalized with activin in the presence of the BMP inhibitor LDN-193189 / DM3189 (250 nM) in CDM2 for 48 hours to obtain definitive endoderm. The cells are again separated and the differentiation state is again measured in a quantitative manner using pathway analysis for all mentioned signaling pathways. If confirmed, the cells are re-seeded and cultured on the surface material described herein functionalized with a 15 / 15 / 35 / 35 mixture (10 micrograms / ml) of BMP4, FGF2, high Wnt ligand (Wnt3A) and Notch ligand (e.g. Delta) in CDM2 medium containing retinoic acid (2 μM, Sigma) and BMP inhibitor DM3189 (250 nM), optionally supplemented with the differentiation agent methoxy-substituted alkylglycerol-1-0-(2-methoxy)hexadecylglycerol (MHG) for 4 days. The medium is refreshed every 24 hours. Finally, the cells are separated from the surface and pathway analysis is performed once more to confirm that the required final differentiation step has been obtained and the end result (e.g. active Wnt and Notch pathways) can be quantified.

[0113] [Example of surface functionalization with cell receptor ligands] Experiments designed as proof of principle for binding cell receptor ligands to functionalized surfaces are described using TGF-β as a representative example. TGF-β was chosen because the effect of TGF-β can be shown by adding it to the culture medium as well, allowing control experiments of the effect of TGF-β on the cultured cells used. The described method offers a major advantage over currently used culture methods when coating the functionalized material with Hedgehog (HH) or Notch ligands, but control experiments with these ligands were not possible, since these ligands act only if and when they are preferably attached to the surface. However, if coating of functionalized surfaces with TGF-β successfully leads to the induction of TGF-β pathway activity in attached cells, it can be safely concluded that coating with other protein ligands will be similarly effective. TGFbl was purchased from STEMCELL TECHNOLOGIES INC. The TGF-β coating experiments were carried out as follows. WPMY1 fibroblasts were seeded and cultured (DMEM medium with 10% FBS) on functionalized surfaces (SIL-IM-F 0.5%, silicone substrate with linoleic acid as described herein and in the abovementioned international patent application PCT / EP2018 / 068318). The surfaces were coated with TGFbl by incubation with 10 μg of TGFbl (SIL-IM-F 0.5%-TGFbl) according to the described protocol, or as a control non-functionalized (SIL-IM-NF) surface, also incubated with TGFbl (SIL-IM-NF 0.5%). In another control situation, WPMY1 cells were cultured on standard tissue culture plastic and 1 ng / ml of TGFbl was added to the medium. The cells were cultured for 48 hours, released from the surface and signaling pathway analysis was performed on RNA isolated from the cells as described herein and in the above-referenced patent application in the context of determining the activity of signaling pathways. Cells attached and grew on all surfaces.The response to TGF-β was measured as a higher TGF-β pathway score in standard cultures (on culture plastic) compared to unstimulated WPMY1 cells (52% higher), which served as a control for responsiveness to stimulation with TGFbl (Table 1). TGFbl coating of the functionalized surface resulted in a 103% increase in TGF-β pathway activity, while TGFbl coating of the non-functionalized surface resulted in only a 67% increase in TGF-β activity (see Table 1).

[0114] [Table 1]

[0115] This table shows that the functionalized surfaces, when coated with TGFbl, are more effective at stimulating the TGF-β pathway in WPMY1 cells in response to TGF-β ligands. Consistent with this, cell morphology was altered compared to cells on surfaces not coated with TGFbl (see Figure 2).

[0116] In an independent cell culture experiment, tissue culture treated plastic surfaces, non-functionalized surfaces and functionalized surfaces were incubated with different concentrations of TGFbl, 0 ng / ml, 10 ng / ml and 1000 ng / ml (Figure 3, shown as No, 10 ng and 1 microgram; y-axis is TGF-β pathway activity score) and analyzed for TGF-β pathway activity. TGF-β pathway activity increased with the concentration of TGFbl with which the functionalized surfaces were incubated to allow TGFbl to bind to the COOH groups of the functionalized surfaces. This increase was comparable to the effect seen when cells were treated with 1 ng / ml TGFbl in the medium when cultured on tissue culture treated plastic. In contrast to non-functionalized surfaces, a similar incubation with TGFbl did not increase TGF-β pathway activity (Figure 3).

[0117] [Examples of surface functionalization with other cell receptor ligands] Sample preparation: As described in PCT patent application WO2019 / 015988, the carboxyl groups on the polymer surface can be reacted with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, compound II), followed by reaction with N-hydroxysuccinimide (NHS, compound IV), which can react with, for example, the amine groups of a ligand, covalently binding the ligand to the carboxyl groups on the polymer surface. In this way, the ligand can form a scaffold, as disclosed herein. Such scaffolds can be formed with high uniformity in terms of coverage of the surface of a polymer surface, for example, the surface of a membrane body of a fluidic device module, due to the high density and uniform distribution of carboxyl groups across the surface.

[0118] Therefore, in this protocol, before ligand binding to the modified silicone samples, a surface treatment of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS) was performed, combining 0.4M EDC (Sigma-Aldrich) with 0.1M NHS (Sigma-Aldrich). The silicone samples were then submerged in the EDC / NHS solution on a 24-well culture plate at room temperature for 1 hour on a shaker at 300 rpm. After 1 hour, the samples were removed from the solution and placed in a new 24-well culture plate. The samples were then submerged in a solution of the ligand JAG1 (1277-jg, rndsystem) at a concentration of 5ng / mL for 1 hour at room temperature. The samples were then thoroughly washed three times using DH2O. The samples were then placed in CellCrown (Sigma-Aldrich) inserts and placed in a new 24-well culture plate. In parallel, this procedure was performed on Nunc polystyrene well plates and non-modified silicone.

[0119] [Cell culture and extraction] WPMY-1 cells (ATCC) were detached using 0.25% trypsin / EDTA (Thermofisher) and 2.5x10 5A cell suspension of cells was generated using DMEM medium containing 10% FBS, 1% Glutamax and 1% penicillin / streptomycin. Then, 400 μl of the cell suspension (1.0×10 5 1000 x 100 cells) were placed in all samples (including controls). Each well was then filled with 1 mL of DMEM medium. The well plate was then placed in an incubator (37°C / 5% CO2) for 48 hours. RNA was extracted using the RNeasy mini plus kit (Qiagen).

[0120] Pathway analysis: The Philips Pathway Analysis (Pathway Analysis disclosed herein) can be used to determine the activity of cell signaling pathways in human cancer tissues. The test is usually based on RT-qPCR expression analysis of multiple target genes for different pathways (AR, ER, AP1 / MAPK, FOXO, TOBβ, HH, Notch). A pathway activity score will be calculated based on mRNA expression values ​​using a computational model. The RNA of the sample is amplified using a one-step RT-qPCR kit. The extracted RNA sample is used for RT-qPCR amplification in Multi-Pathway-Plate (Philips). Thus, the PCR sample is mixed with PCR reagents and dispensed into a PCR plate containing primer sets pre-spotted for pathway target genes. After running the PCR program, the Cq value (expression value) is used as input to calculate a pathway activity score using pathway calculation software (Philips Pathway Analysis disclosed herein). This methodology was used in this experiment to infer Notch pathway activity (also called pathway activity score).

[0121] Results: Figure 4 shows (from left to right) the results of ligand-induced Notch pathway activation in polystyrene (PS) (left), modified silicone (MS) (middle) and regular silicone (S) (right) culture plates. The NOTCH ligand-conjugated (coated) polystyrene culture plate (left) serves as a positive control, and the unmodified silicone culture plate (right) serves as a negative control. Each ligand-coated / conjugated experiment (right bar in each of three pairs) is accompanied by a corresponding experiment without ligand (left bar in each of three pairs). Pathway activity is shown as a pathway activity score, with higher scores indicating higher Notch pathway activity. As can be seen in Figure 4, upregulation of the Notch pathway was seen in the positive control (polystyrene well plate coated with NOTCH ligand) as well as in the modified silicone samples with the NOTCH ligand conjugated, but not in the no-ligand control samples. No upregulation of Notch pathway activity was seen in the negative control (unmodified silicone) with or without the NOTCH ligand coating procedure.

[0122] Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims.

[0123] In the claims, the word "comprising" does not exclude other elements or steps, and the singular does not exclude a plurality.

[0124] A single unit or device may fulfill the functions of several items recited in the claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

[0125] The computer program may be stored / distributed on a suitable medium, such as an optical storage medium or a solid-state medium, supplied together with or as part of other hardware, but may also be distributed in other forms, such as via the Internet or other wired or wireless communication systems.

[0126] Any reference signs in the claims should not be construed as limiting the scope.

Claims

1. A substrate or scaffold for inducing cells in a predetermined direction of cell function, the substrate or scaffold having an elastomeric material having one or more cell function inducing stimuli attached thereto.

2. 2. The substrate or scaffold of claim 1, wherein inducing the cells comprises or consists of inducing the cells in a predetermined direction of cell differentiation, and the cell function inducing stimulus comprises or consists of a cell differentiation inducing stimulus.

3. 3. The substrate or scaffold of claim 1 or claim 2, wherein the one or more cell function-inducing stimuli are selected from the group consisting of cell receptor ligands, extracellular matrix molecules, artificial receptor agonists or antagonists, and specific antigens for T or B cell receptors.

4. 4. The substrate or scaffold according to any one of claims 1 to 3, wherein the cell function inducing stimulus comprises or consists of a cell receptor ligand, which is for activating signal transduction via a signal transduction pathway selected from the group consisting of nuclear receptors such as ER, AR; progesterone receptor; growth factor pathways such as PI3K-FOXO, JAK-STAT3, MAPK-AP1 pathways; immune pathways such as STAT1 / 2 type I interferon (STAT1 / 2-1), STAT1 / 2 type II interferon (STAT1 / 2-2) pathways; developmental pathways such as Hedgehog, Notch, TGF-β and Wnt signaling pathways; and inflammatory pathways such as NFkB.

5. 5. The substrate or scaffold of claim 1 , wherein the elastomeric material comprises or consists of one or more polysiloxane polymers or polybutadiene polymers.

6. 6. The substrate or scaffold of claim 1 , wherein the one or more cell function inducing stimuli bound to the elastomeric material are obtained by reacting functional groups of the one or more cell function inducing stimuli with other reactive groups of a precursor elastomeric material using a cross-linking reaction selected from the group of bioconjugation reactions.

7. 7. The substrate or scaffold of claim 1 , wherein the one or more cell function inducing stimuli are attached to the elastomeric material via one or more of an amide, amine and ester group.

8. A culture unit comprising a cell culture chamber having therein a substrate or scaffold according to any one of claims 1 to 7.

9. 9. Use of a substrate or scaffold according to any one of claims 1 to 8, or a culture unit according to claim 8, for generating a cell cultured tissue or organ.

10. 1. An in vitro method for generating a cell culture, tissue or organ, said in vitro method comprising: (a) determining first culture conditions controlling an activity of at least one first signal transduction pathway of a cell to be cultured, the activity of the at least one first signal transduction pathway being a selected activity for directing the cell in a first predetermined direction of cellular function; (b) culturing the cells in the culture unit under the first culture conditions to obtain a cell culture; The in vitro method comprising:

11. (c) inferring the activity of the at least one first signaling pathway in a cell sample obtained from the cell culture; and (d) comparing the predicted activity with the selected activity; and, optionally, (e) determining second culture conditions based on the comparison of the estimated activity and the selected activity; 11. The in vitro method of claim 10, further comprising:

12. 12. The in vitro method of claim 11, further comprising determining second culture conditions based on the comparison of the estimated activity and the selected activity, the second culture conditions being determined to (i) reduce deviation of the estimated activity from the selected activity, or (ii) control a selected activity of at least one second signaling pathway distinct from the at least one first signaling pathway, the selected activity of the at least one second signaling pathway being selected to direct the cell in a second predetermined direction of cellular function distinct from the first predetermined direction of cellular function.

13. (f) culturing the cells in the culture unit under the second culture conditions; (g) repeating steps (c) and (d), and, optionally, (h) determining third culture conditions based on a comparison of the activity estimated in step (g) with the selected activity; 13. The in vitro method according to any one of claims 10 to 12, comprising:

14. 14. An in vitro method according to any one of claims 9 to 13, wherein the cellular functions are selected from the group consisting of cell differentiation, maintenance of pluripotency and cell division, migration and metabolism, chemotaxis, adhesion, apoptosis, production of oxidative stress, DNA repair, production of secreted proteins, production of extracellular matrix proteins, preferably cell differentiation is selected.

15. the first and / or second culture conditions are defined by controlled exposure of the cells to one or more cell function-inducing stimuli selected from the group consisting of cell receptor ligands, extracellular matrix molecules, artificial receptor agonists or antagonists, and specific antigens for T or B cell receptors, which stimuli control the activity of the at least one first and / or second signaling pathway; and / or The one or more cell function inducing stimuli are provided in the form of one or more culture units according to claim 8.

15. An in vitro method according to any one of claims 9 to 14.

16. 16. The in vitro method according to any one of claims 9 to 15, wherein the cell is a mouse stem cell, a human stem cell, a pluripotent stem cell, a mesenchymal stem cell, an embryonic stem cell, an adult stem cell, a tumor cell, a cell line.

17. A non-transitory storage medium having stored thereon instructions executable by a digital processing device to perform the in vitro method of any one of claims 9 to 16.

18. A computer program comprising program code means which, when executed on a digital processing apparatus, causes the digital processing apparatus to carry out an in vitro method according to any one of claims 9 to 16.

19. A system for carrying out the in vitro method according to any one of claims 9 to 16, comprising: (a) one or more incubation units; (b) a sensor unit for inferring the activity of at least one first signaling pathway; (c) a controller that compares the estimated activity of the first signaling pathway with a predetermined activity, the predetermined activity being selected to controllably steer the cell in a first predetermined direction of cellular function; and, optionally, (d) a non-transitory storage medium according to claim 13 or a computer program according to claim 14, Preferably, the controller further determines culture conditions based on a comparison of the estimated activity and the predetermined activity, the culture conditions being optionally determined to (i) reduce deviation of the estimated activity from the predetermined activity, or (ii) control activity of the at least one second signaling pathway. system.

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