WO2021230750A1

WO2021230750A1 - Screening plate and method for co-culturing cells

Info

Publication number: WO2021230750A1
Application number: PCT/NL2021/050318
Authority: WO
Inventors: Gilles Philippus Van Wezel; Dino VAN DISSEL
Original assignee: Universiteit Leiden
Priority date: 2020-05-15
Filing date: 2021-05-17
Publication date: 2021-11-18

Abstract

A screening plate has a plurality of wells for culturing cells therein and comprises a first layer defining top well sections of the plurality of wells, and a second layer defining bottom well sections of the plurality of wells. A membrane is provided between the first layer and second layer and divides the plurality of wells into top well sections above the membrane for holding a first cell culture medium with cells and bottom well sections below the membrane for holding a second cell culture medium with cells. The bottom well section and top well section of each well are in fluid communication with each other through the membrane.

Description

Title: Screening plate and method for co-culturing cells

FIELD OF THE INVENTION

The invention relates to a screening plate and a method for co- culturing cells.

BACKGROUND

In medicine development there is a continued demand for novel compounds, e.g. molecules, that can be suitably used in or applied as new medicine, as existing medicine may have serious side effects and/or become less effective due to a growing resistance thereto of the subject treated (Cooper and Shlaes, 2011; Lewis, 2013). Natural products derived from microorganisms provide a relevant source of potential novel compounds that can be employed in medicine and agriculture. Among the organisms that are capable of producing interesting natural products to this end, the Gram- positive bacteria Actinomycetes, and especially the genus Streptomyces, have gained high levels of attention. Actinomycetes are a diverse family of filamentous bacteria that are known to produce a wealth of useful metabolites, such as antibiotics, anticancer, antifungal, and immunosuppressant compounds (Barka et al., 2016; Berdy, 2005). Sequencing of Streptomyces genomes established the presence of silent biosynthetic gene clusters (BGCs) for natural products, implying that the potential of these bacteria as producers of bioactive molecules is likely much larger than originally anticipated (Challis and Hop wood, 2003; Medema et al., 2015; Nett et al., 2009).

Co-cultivation of Actinomycetes with other organisms has shown that cryptic gene clusters in the Actinomycetes, which normally are not activated when growing under single-strain laboratory conditions, can be elicited to expression by elicitor compounds secreted by the co-cultivated organism (van der Meij et al., 2017; Wu et al., 2015). Co-cultivation of Actinomycetes with other organisms thus may be employed to screen for novel bioactive molecules such as antibiotics produced by Actinomycetes, as well as for elicitor compounds secreted by the co-cultivated organism.

Conventional screening methods for testing microorganisms (producer), such as bacterial strains or yeasts, for their production of bioactive agents or molecules generally includes growing the producer in or on a solid or a liquid culture medium followed by assessing the composition of the culture medium for its bioactivity on a target receiver cell line (Baltz, 2008; Lewis, 2013). For example, a known method for antibiotic screening of Actinomycetes involves cultivating Actinomycetes on medium containing plates followed by overlaying with a soft agar containing a target cell population, such as a different bacterial strain, incubating for a period of time and analyze antibacterial activity, e.g. by measuring a zone of inhibition.

This known antibiotic screening method is however not very suitable for screening for novel compounds with bioactivity against eukaryotic cells as target receiver cell line, such as compounds with antitumor activity, due to contamination problems and particularly unfavorable growth conditions, i.e. cytotoxic conditions, limiting or preventing co-cultivation of the producer with eukaryotic cells, e.g. cancer cells.

It is accordingly an aim of the invention to provide an efficient and reliable screening plate and screening method that enables screening of non- mammalian cells, in particular bacterial cells, co-cultivated with mammalian cells for production of bioactive compounds that modulate growth of the co-cultivated mammalian cells. It is a further aim of the invention to provide an efficient and reliable screening plate and method that enables screening of mammalian cells for bioactive elicitor compounds that activate the expression of bio-synthetic gene clusters encoding for growth-manipulating compounds in bacterial cells, particularly Actinomycetes, and especially of the genus Streptomyces. Particularly, it is an aim to provide such screening plates and screening methods arranged for medium-throughput assays or high-throughput assays of bioactive compounds produced by non- mammalian cells that modulate growth of the co-cultivated mammalian cells.

DESCRIPTION OF THE INVENTION

Accordingly, in a first aspect there is provided a screening plate having a plurality of wells for culturing cells therein. The plate comprises a first layer defining bottom well sections of the plurality of wells, and a second layer defining top well sections of the plurality of wells. The screening plate further comprises a membrane between the first layer and second layer which membrane divides the plurality of wells into the bottom well sections below the membrane for holding a first cell culture medium with cells and the top well sections above the membrane for holding a second cell culture medium with cells. The bottom well section and upper well section of each well are in fluid communication with each other through the membrane.

The top and bottom well sections may be provided with cells of different cell populations and are physically separated from each other by the membrane, which prevents migration of cells from the top well section into the bottom well section and vice versa. The membrane is further arranged to allow exchange of compounds between the cell populations cultivated in the top well section and the bottom well section. Particularly the membrane may be arranged to allow exchange of growth-modulating compounds, such as antibiotic compounds, and/or eliciting compounds. The screening device thus enables back-to-back co-cultivation of different cell populations, which remain physically separated, while allowing the different cell populations to communicate with each other via compound exchanges. Growth conditions on either side of the membrane, i.e. in the top and bottom well sections, may be optimized for an associated cell population that is intended to be cultivated therein.

The membrane may consist of a single membrane layer or comprise multiple membrane layers, i.e. two or more membrane layers. Each membrane layer of the multiple membrane layers may be the same or may be different. In particular the membrane may comprise at least two different membrane layers. The different membrane layers may provide different filtering properties. For example the membrane may comprise a first membrane layer arranged to prevent migration of cells from the top well section into the bottom well section and vice versa and a second membrane layer arranged to provide selective exchange of growth- modulating compounds, such as antibiotic compounds, and/or eliciting compounds. The membrane layers may for instance differ in material from which the membranes are formed or may be formed from the same material. The membrane layers may also differ in thickness, density, permeability, pore size, and/or other features that affect the filter properties of the membrane layer concerned. For example the membrane at least comprises a semipermeable membrane layer arranged to prevent migration of cells from the top well section into the bottom well section and vice versa and optionally comprises an additional membrane layer selective for certain classes of compounds, e.g. molecules. The additional membrane layer may for instance consist of or comprise a layer of PVDF or Hybond-P arranged to prevent passage of proteinaceous and/or hydrophobic compounds. The membrane layers may comprise a woven or non-woven. The membrane layers may be flexible, for example a textile or fiber-based membrane layer, or relatively rigid, e.g. a plastic or metal mesh type membrane layer. The membrane may comprise the membrane layers directly overlaying each other, i.e. with no free space between the layers, and/or with membrane layers separated, i.e. with some space between the layers. In a particular aspect the top well section may be provided with a liquid culture medium suited for culturing mammalian cells, while the bottom well section may be provided with a substantially solid culture medium, such as an agar based culture medium, suited for culturing producer cells such as bacterial cells, e.g. Actinomycetes, or yeast cells. After co-culturing two cell populations for a period of time, cells from either or both populations may be easily extracted from their respective well sections in the screening plate wells for further research. Optionally one of the cell populations may be conveniently replaced or reseeded, while the other cell population, separated by the membrane, can remain substantially undisturbed.

In an aspect, the screening plate may be provided with a first bacterial strain in the top well sections, and a second bacterial strain in the bottom well sections. Compounds excreted by the first bacterial strain may affect the second bacterial strain and vice versa. For instance, the first bacterial strain may produce elicitor compounds that diffuse across the membrane, which may be arranged for this purpose, into the bottom well section. Those elicitor compounds may affect the second bacterial strain in the bottom well section in such a way that otherwise silent bio-synthetic gene clusters are activated in the second bacterial strain. These bio- synthetic gene clusters may for instance encode for antibiotic compounds which have growth-inhibiting properties. Those antibiotic compounds in turn can optionally diffuse across the membrane, which may be arranged for this purpose, to the top well section. The screening plate allows convenient extraction of the culture media in the wells for determining elicitor compounds and/or growth-modulating compounds therein.

The plurality of wells of the screening plate allows for at least medium-throughput and particularly high-throughput screening, for example with various cell populations under various conditions. The multi- well plate may comprise for example a standard number of 12, 24 or 96 wells, or any other plural number of wells, although at least 12 wells is preferred. Each of the plurality of wells may have a substantially cylindrical shape or any other suitable shape. For example each well may be formed by a circumferential wall defining a well space for holding the cells and/or media, which circumferential wall may for instance be cylindrical to define a well space with a circular cross section. The wells may be positioned within the screening plate at a distance to each other or adjacent to each other. For example the circumferential walls of two consecutive wells in the screening plate may abut or may be distanced from each other by an intermediate space between the wells. The wells may be open at a top and/or bottom side of the screening plate to allow for visual inspection of the cell cultures in the wells. Moreover, the first and second layer of the plate may be made of a substantially inert and transparent material. For example polytetrafluoroethylene (Teflon) may be used as an autoclavable material for convenient sterilization of the plate. Alternatively, or in addition, glass may be used to make either or both of the first and second layer of the plate.

A permeability of the membrane or membrane layer may be arranged, for example by adapting a pore size of the membrane, to allow diffusion of compounds with a specific molecular size or mass while preventing larger compounds or cells from migrating through the membrane. The membrane may further be provided as a single layer interposed between the first and second layer of the screening plate for dividing each of the plurality of wells, or at least a majority of the wells, into a top well section and a bottom well section. The screening plate may be assembled by sandwiching the membrane between the first and second layer.

Optionally, a sealing means is provided between the first layer and the second layer for liquid tight sealing of the bottom well section to the top well section of each of the plurality of wells. The sealing means ensures that culture media and cells remain in the wells, and prevents that culture media and cells can leak into any intermediate space between the first and second layer. This prevents or at least limits contamination between a plurality of wells. The sealing means further prevents or at least limits contamination of the wells from an outside environment exterior to the screening plate.

Optionally, a first sealing layer is provided between the membrane and the first layer, and a second sealing layer is provided between the membrane and the second layer.

Optionally, an O-ring or other radial seal body is provided circumferentially around each of the plurality of wells, which O-ring or other radial seal body is provided between the membrane and the first layer and/or between the membrane layer and the second layer. The O-ring or other radial seal body allows for an enhanced localized radial sealing of each of the plurality of wells, to prevent an exchange of fluids between different wells via any intermediate space between the first layer and the second layer. Specifically, the O-ring or other radial seal body may be provided between the first layer and the first sealing layer, and/or between the second layer and the second sealing layer.

Optionally, the plate comprises coupling means for coupling the first layer and second layer to each other with the membrane interposed. This makes the screening plate easy to handle in use while providing a reliable connection between the different layers of the plate. The screening plate may for example be assembled by providing the membrane, and optionally a sealing layer, between the first and second layers, and mechanically coupling the first layer and the second layer with each other by means of the coupling means. Similarly, the screening plate may be disassembled by removal of the coupling means, which allows for easy replacement and cleaning of the components, e.g. layers and sealing means, of the screening plate. In particular, the membrane or at least one membrane layer may be interchanged for another membrane or membrane layer having properties as desired, for example suitable for a particular screening experiment. The coupling means may comprise a relative simple screw coupling, for example by means of a nut and bolt, for coupling the first and second layer of the plate to each other.

Optionally, the coupling means comprise clamping means for exerting a clamping force on the first layer and second layer for reliable liquid tight sealing the bottom well section and top well section of each well and the interposed membrane to each other. Clamping enhances and maintains a sealing between the first and second layer. In case the screening plate is provided with an O-ring or other radial seal body around each of the wells, sealing is particularly focused at the locations of the O- rings or other radial seal bodies around each of the wells.

Optionally, the plate comprises a first substantially rigid force distribution plate and a second substantially rigid force distribution plate, wherein the first layer and second layer are provided between the first distribution plate and second distribution plate. The distribution plates provide an even distribution of a clamping force over an outer surface of the first layer and second layer. Any one of the distribution plates may for example be formed as a flat, sheet-like, body and/or may be made of a rigid and durable material, for example a metal such as stainless steel.

Optionally, the plate comprises a removable cover for covering the plurality of wells. The removable cover prevents contamination from outside of the screening plate when placed over the wells, while allowing easy access to the wells once removed. A size of the screening plate may be adapted to be compatible with removable covers of conventional multi-well plates, such as a of conventional 96 wells plate, in order to allow use thereof on the screening plate. Alternatively or additionally the screening plate may be provided with a removable cover that does not fit on conventional multi-well plates in order to prevent possible confusion or misuse. Optionally, the membrane comprises a Hybond-N membrane or membrane layer. A Hybond-N membrane provides an optimal balance between permeability on the one hand, while allowing retaining of liquid culture medium within a well section. This is particularly advantageous in a vertical setup, i.e. wherein the top well section is provided with a liquid culture medium and the bottom well section is provided with a substantially solid culture medium. In such a setup, the liquid culture medium may leak through the membrane and collect in the bottom well section, resulting in a depletion of the top well section. This poses a restriction to a maximum time-scale of an experiment. A Hybond-N membrane allows for effective compound exchange between the top and bottom well sections, while minimizing leakage of the liquid culture medium through the membrane from the top well section to the bottom well section. The Hybond-N membrane may comprise the Hybond-N membrane layer in addition to a further membrane layer arranged to prevent migration of cells from the top well section into the bottom well section and vice versa.

Alternatively, or in addition, the membrane comprises a polycarbonate based membrane or membrane layer, and preferably comprises a polycarbonate Track-Etched (PCTE) membrane or membrane layer. This minims or completely prevents cells larger than the pore size of the membrane of passing the membrane from the top well section into the bottom well section and vice versa.

Optionally, the membrane is provided with a coating, for example a coating comprising poly-L-lysine, and preferably a fibronectin coating, arranged for adhering cells to the membrane. This facilitates cell growth on a surface of the membrane for cells that require an attachment surface for growth, and further allows for growth in close range to a cell culture on the opposite side of the membrane. Optionally, the membrane is provided with a fibronectin coating density of about 1 μg/cm² - 4 μg/cm² preferably about 2 pg/cm². Such density is found to provide a successful adhesion of cells to the membrane.

Optionally, the bottom well section is arranged to hold, at least in use of the plate, a substantially solid cell culture medium adjacent the membrane. Several cell lines, in particular several yeast cell lines or bacterial cell lines such as Actinomycetes, are preferably grown in or on substantially solid culture media such as jelly-like culture media, e.g. agar. Particularly, the production of bioactive compounds in Actinomycetes may be tied to the morphological development of the cell population which is facilitated by a substantially solid culture medium.

Optionally, the bottom well section below the solid cell culture medium comprises a head space. Several cell lines, particularly Actinomycetes, produce significantly more molecules when grown on a substantially solid culture medium with a headspace, than in submerged cultures. Many compounds are not properly expressed in when cultured in submerged liquid culture media.

Optionally, the culture medium comprises a concentration of SFM agar of approximately 2%-6% in particular about 4%. It was found that an overall liquid retention of an agar gel greatly depends on a concentration of agar in the gel. For example, a gel dehydrates slower with higher percentage of agar in the gel. Liquid retention of the substantially solid gel is desirable to maintain contact with walls of a well to minimize leakage of liquid culture medium from the top well to the bottom well section. However, a high liquid retention results in a lower diffusion of other molecules. Accordingly, is was found that an agar concentration of approximately 2%-6%, and particularly an agar concentration of about 4%, gives an optimal balance between liquid retention and leakage on the one hand, and diffusion rates in the substantially solid culture medium on the other hand. Optionally, the cell culture medium comprises glycerol. Glycerol protects bacteria from heat stress when incubating at a temperature of about 37°C. By providing glycerol, for example in a concentration of about 1%, in the culture medium, bacteria and mammalian cells can be co- cultured in the screening plate at a temperature of about 37°C.

Optionally, the bottom well section is arranged to hold, at least in use of the plate, a cell culture of Actinomycetes. As a consequence of their filamentous lifestyle, many Actinomycetes are hard to cultivate in small- scale liquid-grown cultures such as microtiter plates, due to the formation of large mycelial clumps, high viscosity and slow growth. Furthermore, the production of bioactive compounds is typically tied to morphological development, and many compounds are therefore not properly expressed in submerged cultures. Accordingly, the bottom well section arranged to hold Actinomycetes particularly comprises a solid cell culture medium suitable for growing and/or maintaining or cultivating Actinomycetes.

In a second aspect of the invention, there is provided a method for screening for elicitor compounds/growth modulating compounds comprising co-culturing a first cell culture of growth- modulating compounds producing cells, preferably yeast cells or bacterial cells, more preferably Actinomycetes, with a second cell culture of cells of interest, preferably mammalian cells, more preferably cancer cells. The first cell culture and second cell culture are kept separated from each other for direct physical contact via a semi-permeable membrane that allows exchange of compounds secreted by cells between the first cell culture and the second cell culture. The growth-modulating compounds may for example be cytotoxic compounds. The second cell culture of cells of interest may for example be a bacterial cell line, particularly a pathogenic bacterial cell line, that is capable of producing eliciting compounds for eliciting the production of antibiotics in the first cell culture that inhibit the growth of such bacteria of the second cell culture. Optionally, the second cell culture is grown and/or maintained in a substantially liquid cell culture medium. Certain cell populations, in particular, mammalian cells are preferably cultivated in liquid culture medium.

Optionally, the first cell culture is grown in a substantially solid culture medium, preferably an agar based culture medium. Certain cell populations, in particular bacterial cells such as Actinomycetes, are preferably grown on substantially solid culture media. For example, as a consequence of their filamentous lifestyle, many Actinomycetes are hard to cultivate in small-scale liquid-grown cultures such as microtitre plates, due to the formation of large mycelial clumps, high viscosity and slow growth. Furthermore, the production of bioactive compounds may be tied to morphological development of the bacterial cell population, and many compounds are therefore not properly expressed in submerged liquid cultures.

Optionally, the method comprises, after co-culturing the first and second cell cultures, determining a composition of the first and/or second culture medium. The composition of the culture medium may be determined by means of Nuclear Magnetic Resonance (NMR) spectroscopy, and/or Mass Spectroscopy (MS) such as Liquid Chromatography-Mass Spectroscopy (LS- MS). The composition of the first and/or second culture medium may reveal excreted bioactive compounds by the co-cultured cell cultures.

Optionally, the composition of the first and/or second culture medium is compared with a reference composition. The reference composition may for example comprise a composition of culture medium originating from a cell culture which has been cultured in isolation, i.e. in absence of a different cell culture. For example, a first reference composition may be a composition of a culture medium in which only the cells of interest, such as mammalian cells, more specifically cancer cells have been cultured. In another example, a second reference composition may be a composition of a culture medium in which only the culture of growth-modulating compounds producing cells, such as yeast cells or bacterial cells, more specifically fungi or Actinomycetes, have been cultured. By comparing the first culture medium and/or the second culture medium with the first and/or second reference composition, a composition difference can be determined between co-cultured and isolated cultures. This allows for the identification of compounds in the composition of the first and/or second culture medium that are a result of interaction between the cells of interest and the growth- modulating compound producing cells. Optionally, eliciting compounds are identified in the composition of the culture medium. A comparison between the composition of the first and/or second culture medium with the first and/or second reference composition, allows to identify elicitor compounds which may have been produced by the cells of interest, for instance cancer cells, in co-culture with the growth- modulating compound producing cells, for instance

Actinomycetes. Similarly, a comparison between the composition of the first and/or second culture medium with the first and/or second reference composition, allows to identify growth-modulating compounds which may have been produced by the growth- modulating compound producing cells, for instance Actinomycetes, in co-culture with the cells of interest, for instance cancer cells.

Optionally, the eliciting compounds are isolated from the first and/or second culture medium.

Optionally, the growth-modulating compounds are isolated from the first and/or second culture medium.

Optionally, growth-modulating compounds will be assessed for their ability to promote growth of a reference cell line. A reference cell line may for example be a cancer cell line, or a cell line of Chinese Hamster Ovary cells. Optionally, growth-modulating compounds will be assessed for their ability to inhibit growth of a reference cell line particular cancer cells, such as pancreatic cancer cells.

Optionally, an elicitor compound is assessed for its ability to activate an expression of (silent) bio- synthetic gene cluster in a reference cell line, which bio-synthetic gene cluster specifies for a growth-modulating compound. Here are reference cell line may be a cell line of non-mammalian cells, such as bacterial cells, in particular Actinomycetes.

BRIEF DESCRIPTION OF THE FIGURES

These and other aspects of the present invention are hereinafter further elucidated by the appended drawing with figures and the corresponding embodiments, which form part of the present application. The drawing is not in any way meant to reflect a limitation of the scope of the invention, unless this is clearly and explicitly indicated. In the drawing shows:

Fig. 1 an exploded view of a screening plate in an aspect of the invention;

Fig. 2 a screening of bacterial strains showing the production of cytotoxic metabolites on different solid media using a Sandwich Plate screening protocol. In all wells, the top compartment contained HeLa cells. Bars indicate the fluorescence level relative to the control ± SD (n=3 for strains, n=2 for control), as measured using the resazurin fluorescence assay. The control describes wells that were not inoculated with bacteria;

Fig. 3 a screening of Streptomyces sp. QL37, (QL37) variants for production of cytotoxic metabolites employing the Sandwich Plate screening protocol. Three media were screened with and without HeLa cells in the top compartment. Bars indicate relative fluorescence normalized to the control wells without bacteria ± SD (n=3 for strains, n=2 for control). Asterisks indicate values that differed significantly between HeLa cells present and not present (t-test, a = 0.05). The arrow indicates a value that was repeated due to very high SD;

Fig. 4 a LC-MS analysis of supernatants obtained by culturing bacterial strains QL37, QL37 mutated for lugOV (QL3ΔlugOV), and Streptomyces coelicolor M1152 + lug (the lug gene cluster encodes the production of angucyclines and lugdunomycin (Wu et al., 2019)) on ISP4 medium in the Sandwich plates showing a Chromatogram overlay of all strains;

Fig. 5 a LC-MS analysis of supernatants obtained by culturing bacterial strains (QL37, QL3ΔlugOV, M1152+ lug) on ISP4 medium in the Sandwich plates showing a mass spectrum of the 353.1023 peak identified in fig. 4. The mass of the molecule corresponds to an unrearranged benz-[a]- anthracene angucycline, shown on the spectrum.

DETAILED DESCRIPTION OF THE FIGURES

Figure 1 shows an exploded view of a screening plate 1 comprising a plurality of wells for culturing cells. In Figure 1, the screening plate 1 comprises an array of 24 wells. The screening plate 1 comprises a first layer 3 and a second layer 5. The first layer 3 defines a plurality of top well sections, and the second layer defines a plurality of bottom well sections. In particular, the first layer defines 24 top well sections and second layer defines 24 bottom well sections. Each of the 24 top well sections is associated with a respective bottom well section for forming the wells, wherein the wells are physically and fluidly separated from one another to avoid communication between any of the wells. The first and second layers 3,5 are each made of a substantially inert material. In particular, the first and second layers 3,5 are each made of Teflon.

Interposed between the first layer 3 and the second layer 5 is a membrane 7. The membrane 7 is formed as a single layer which divides each of the plurality of wells, into respective top well sections and bottom well sections. Cell cultures may be provided in top well sections and in the bottom well sections, wherein the membrane 7 prevents migration of cells from any top well section to any bottom well section and vice versa. The membrane 7 is further arranged to provide fluid communication between associated top well sections and bottom well section, such that bioactive compounds may be interchanged between the associated well sections. This allows for back-to-back co-cultivation of cell cultures, wherein cell populations in a well on opposite sides of the membrane can communicate through the membrane 7, while remaining physically separated.

To prevent or at least minimize leakage of culture medium and cells between wells, the screening plate 1 is provided with sealing means. In particular, the sealing means comprise first sealing layer 9 interposed between first layer 3 and the membrane 7, and a second sealing layer 11 interposed between the second layer 5 and the membrane 7. The first 9 and second 11 sealing layers are each formed by a resilient material such as silicone rubber. The first and second sealing layers 9,11 are each provided with a plurality of through holes, each through hole associated to the wells of the screen plate 1. The shape of each of the through holes closely resembles a cross-sectional shape of an associated well or associated well section to provide a sealing around each of the wells.

Coupling means are provided to couple the layers of the screening plate to each other. In the example, the coupling means comprise one ore more screws or bolts 13 for aligning the layers of the plate 1 and for coupling the layers of the screening plate to each other by means of a screw coupling. To this end each of the layers, e.g., the first layer 3, second layer 5, membrane 7, first sealing layer 9, second sealing layer 11, comprise through holes for receiving one or more screws or bolts to align the layers and couple the layers to each other. The layers of the plate 1 may also be decoupled by removing the one or more bolts 13. This way, the layers can be easily cleaned or replaced. It is envisioned that there are other coupling means suitable for coupling and/or decoupling the layers to each other.

Furthermore, clamping means may provided to exert a clamping force on the screening plate 1, in particular on the layers of the screening plate 1. This could enhance sealing. In this example, the clamping means comprise the one or more screw of bolts 13, wherein the one or more screws or bolts 13 can be tightened to induce a clamping force on the screening plate 1. The clamping means may also comprise additional clamping means. The one or more bolts 13 can be distributed evenly over the screening plate 1, to evenly distribute a clamping force over the screening plate 1. For example, bolts may be provided at each corner of the screening plate. To avoid concentrated loads on the first and second layers 3, 5 of the screening plate 1, which could damage the first and second layers 3, 5, a distribution plate may be provided to distribute a clamping force from the clamping means, e.g. the one or more bolts 13, evenly over the screening plate 1. In Figure 1, the screening plate is provided with a first distribution plate 15 adjacent the first layer 3 at an outward facing side of the first layer 3, and a second distribution plate 17 adjacent the second layer 5 at an outward facing side of the first layer 5. The distribution plates 15, 17 are provided with a plurality of through holes associated with the wells or well sections. The distribution plate is preferably made of relatively stiff material such as a metal like stainless steel.

The screening plate may further be provided with a first and second cover (not shown) to cover the wells on either side of the wells. The screening plate 1 may be adapted to be fitted with covers of conventional multi- well screening plates.

As is shown in the close up view of Fig. 1, the screening plate is further provided with a plurality of O-rings arranged around a perimeter of each of the well sections. A first plurality of O-rings 19 are interposed between the first layer 3 and membrane 7 in particular between the first layer 3 and the first sealing layer 9. A second plurality of O-rings 21 are interposed between the second layer 5 and the membrane 7, in particular between the second layer 5 second sealing layer 11. This way, sealing is focused at the perimeter of the well sections to prevent leakage of culture medium and cells to neighboring well sections.

In an example, a top well section is provided with a liquid culture medium with cancer cells, and a bottom well sections is provided with a substantially solid culture medium with Actinomycetes. This is schematically depicted in Figure 1. It may be preferred to leave a headspace in the top well section and/or bottom well section for optimal growth conditions. For example, when culturing actinomyctes in a substantially solid culture medium, it is preferred to leave a headspace. As such, the bottom well section is not entirely filled with culture medium. Similarly, it may be desired to leave a headspace in the top well section as well.

EXAMPLES

The invention will further be discussed in conjunction with an example of an experimental setup. In the experimental setup, six different bacterial strains were screened on five substantially solid media for production of antitumor compounds. Firstly, the bacterial strains were pre- cultured on the substantially solid media for four days and then co-cultured with HeLa cells in a liquid DMEM culture medium for two days in a screen plate. After co-culturing, the liquid DMEM medium was tested for cytotoxicity compounds against HeLa cells. This was done by using a resazurin fluorescence assay. Results are shown in Figure 2.

Variable cytotoxicity profiles were observed for the tested bacteria. Some bacterial strains, such as S. coelicolor M1152 and Streptomyces sp. QL37AminPKS did not produce a cytotoxic altered DMEM in all the media tested. This contrasts S. peucetius, which produced cytotoxic altered liquid DMEM culture medium when grown on the minimal media and ISP4, but not when grown on MYM or R5+ Peptone + Mannitol.

The Streptomyces sp. QL37 variants showed high variation in their cytotoxicity profiles. The wild type Streptomyces sp. QL37 did not produce a lot of metabolites influencing cellular proliferation in most media, with the lowest relative fluorescence being produced on MM+Man+Gly. Contrasting to this, the Streptomyces sp. QL3ΔlugOV variant produced cytotoxic altered DMEM in all media, especially in ISP4. The S. coelicolor M1152 110 (4)

(M1152 +lug) variant showed similar cytotoxicity to the Streptomyces sp.QL3ΔlugOV variant in most media, except for MM + 0.5 % mannitol + 1% glycerol, where the relative fluorescence was around the level of the control without bacterial inoculate.

The previous screening revealed that the Streptomyces sp. QL37 strains showed high differences in the production of cytotoxic molecules among each other. To further test this, the sandwich plate protocol was used to screen six variants of the Streptomyces sp. QL37 strain on ISP4, MM+ 1 % Glycerol + 1 % Mannitol and R5 + 0.8 % Peptone + 1 % Mannitol for the production of antitumor metabolites. The results are shown in Figure 3. Additionally, it was tested if an observed effect would be due to an eliciting effect from the co-culture with HeLa cells. Thus, all measurements were performed both with and without seeding cells in the top compartment. To evaluate the robustness of the assay, all measurements, even for conditions that had been tested previously, were performed again.

Except for some singular measurements, the results did not differ significantly from the results in the previous figure and were mostly found within two SD of each other. Outlier values include S. coelicolor M1152 110 (4) (M1152 + lug) on R5 + cells, as well as on ISP4 +cells, which would need to be repeated with a higher sample size. Similar to previous results, Streptomyces sp. QL37 wild type (WT) did not produce cytotoxic altered DMEM on most media, except when cultured on R5 + 0.8 % Peptone + 1% Mannitol. Here, in co-culture with HeLa cells, a moderate reduction of fluorescence of approximately 50 % could be observed. For most conditions tested, the Streptomyces sp. QL37ΔlugOII and Streptomyces sp. QL37ΔlugOIII variants did not produce altered DMEM with a significantly lower relative fluorescence than Streptomyces sp. QL37 WT and averaged around 80 %. This contrasts with the Streptomyces sp. QL3ΔlugOV and S. coelicolor M1152 + lug variants, which again produced altered DMEM with low relative fluorescence to the control.

The influence of the presence of HeLa cells in the top compartment on the bacterial strains tested was evaluated by statistical comparison of the values obtained with and without HeLa cells in the top compartment of the Sandwich Plate. Significantly different fluorescence levels with HeLa cells present could be obtained for several values. These included, for example, Streptomyces sp. QL37 on minimal medium or S. coelicolor M1152 +lug on minimal medium. The Streptomyces sp. QL37ΔlugOV mutant showed a significant effect of cells present in all three media.

The previous experiments demonstrated that Streptomyces sp. QL37ΔlugOV and S. coelicolor M1152 110 (4) (M1152 +lug) produced compounds on ISP4 that were cytotoxic to HeLa cells. To gain insight into the metabolic profile of these strains, altered DMEM was produced by inoculating these strains and Streptomyces sp. QL37 on ISP4 in the Sandwich Plate. Liquid DMEM was added on top without cells and after two days the altered DMEM was removed, extracted, then analyzed using high- resolution LC-MS. Results are shown in Fig. 4. One peak was detected to be very prominent both in the Streptomyces sp. QL3ΔlugOV mutant, as well as the M1152 + lug heterologous expression mutant. The mass spectrum of this peak revealed a compound whose mass corresponds to an unrearranged benz [a] -anthracene angucycline (Wu et al., 2019), the structure of which is depicted in Fig. 5. The peak was not detected in the Streptomyces sp. QL37 WT or the control. References

Baltz, R.H. (2008) Renaissance in antibacterial discovery from actinomycetes. Curr Opin Pharmacol 8: 557-563. Barka, E.A., Vatsa, P., Sanchez, L., Gavaut-Vaillant, N., Jacquard, C.,

Meier-Kolthoff, J., Klenk, H.P., Clement, C., Oudouch, Y., and van Wezel, G.P. (2016) Taxonomy, physiology, and natural products of the Actinobacteria. Microbiol Mol Biol Rev 80: 1-43.

Berdy, J. (2005) Bioactive microbial metabolites. J Antibiot (Tokyo) 58: 1-26. Challis, G.L., and Hopwood, D.A. (2003) Synergy and contingency as driving forces for the evolution of multiple secondary metabolite production by Streptomyces species. Proc Natl Acad Sci USA 100: 14555-14561.

Cooper, M.A., and Shlaes, D. (2011) Fix the antibiotics pipeline. Nature 472: 32. Lewis, K. (2013) Platforms for antibiotic discovery. Nat Rev Drug Discov 12: 371-387.

Medema, M.H. et al. (2015) Minimum Information about a Biosynthetic Gene cluster. Nat Chem Biol 11: 625-631.

Nett, M., Ikeda, H., and Moore, B.S. (2009) Genomic basis for natural product biosynthetic diversity in the actinomycetes. Nat Prod Rep 26:

1362-1384. van der Meij, A., Worsley, S.F., Hutchings, M.I., and van Wezel, G.P. (2017) Chemical ecology of antibiotic production by actinomycetes. FEMS Microbiol Rev 41: 392-416. Wu, C., Zacchetti, B., Ram, A.F., van Wezel, G.P., Claessen, D., and Hae

Choi, Y. (2015) Expanding the chemical space for natural products by Aspergillus- Streptomyces co-cultivation and biotransformation. Sci Rep 5: 10868.

Wu, C., van der Heul, H.U., Melnik, A.V., Lubben, J., Dorrestein, P.C., Minnaard, A.J., Choi, Y.H., and van Wezel, G.P. (2019) Lugdunomycin, an Angucycline- Derived Molecule with Unprecedented Chemical Architecture. Angew Chem Int Ed Engl 58: 2809-2814.

Claims

1. Screening plate having a plurality of wells for culturing cells therein, the plate comprising a first layer defining top well sections of the plurality of wells, and a second layer defining bottom well sections of the plurality of wells; and a membrane between the first layer and second layer which membrane divides the plurality of wells into top well sections above the membrane for holding a first cell culture medium with cells and bottom well sections below the membrane for holding a second cell culture medium with cells, wherein the bottom well section and top well section of each well are in fluid communication with each other through the membrane.

2. Screening plate according to claim 1, wherein a sealing means is provided between the first layer and the second layer for liquid tight sealing of the bottom well section to the top well section of each of the plurality of wells.

3. Screening plate according to claim 2, wherein a first sealing layer is provided between the membrane layer and the first layer, and a second sealing layer is provided between the membrane layer and the second layer.

4. Screening plate according to claim 3, wherein, circumferentially around each of the plurality of wells, a sealing O-ring is provided, which O- ring is provided between the membrane layer and the first layer and/or between the membrane layer and the second layer.

5. Screening plate according to any one of the foregoing claims, wherein the plate comprises coupling means for coupling the first layer and second layer to each other with the membrane interposed.

6. Screening plate according to claim 5, wherein the coupling means comprise clamping means for exerting a clamping force on the first layer and second layer for liquid tight sealing the bottom well section and top well section of each well and the interposed membrane to each other.

7. Screening plate according to any one of the foregoing claims, wherein the plate comprises a first substantially rigid distribution plate and a second substantially rigid distribution plate, wherein the first layer and second layer are provided between the first distribution plate and second distribution plate.

8. Screening plate according to any one of the foregoing claims, wherein the plate comprises a removable cover for covering the plurality of wells.

9. Screening plate according to any one of the foregoing claims, wherein the membrane comprises a PCTE membrane.

10. Screening plate according to any one of the foregoing claims, wherein the membrane is provided with a coating, preferably a fibronectin coating, arranged for adhering cells to the membrane.

11. Screening plate according to any one of the foregoing claims, wherein the bottom well section is at least partly filled, at least in use of the plate, with a substantially solid cell culture medium adjacent the membrane.

12. Screening plate according to claim 11, wherein the bottom well section below the solid cell culture medium comprises a head space.

13. Screening plate according to claim 11 or claim 12, wherein the culture medium comprises a concentration of SFM or MM agar containing one or more carbon sources of approximately 4%.

14. Screening plate according to any one of claims 11-13, wherein the cell culture medium comprises glycerol.

15. Screening plate according to any one of the foregoing claims, wherein the bottom well section is arranged to hold, at least in use of the plate, a cell culture of non-mammalian cells.

16. Screening plate according to any one of the foregoing claims, wherein the top well section is arranged to hold, at least in use of the plate a substantially liquid cell culture medium.

17. Screening plate according to claim 16, wherein the substantially liquid cell culture medium is, at least in use of the plate, provided with mammalian cells.

18. Method for screening for elicitor compounds and/or growth modulating compounds comprising co-culturing a first cell culture of growth- modulating compounds producing cells, preferably yeast cells or bacterial cells, more preferably Actinomycetes, with a second cell culture of cells of interest, preferable mammalian cells, more preferably cancer cells, wherein the first cell culture and second cell culture are kept separated from each other for direct physical contact via a semi-permeable membrane that allows exchange of compounds secreted by cells between the first cell culture and the second cell culture.

19. Method according to claim 18, wherein the first cell culture is grown in a substantially solid culture medium, preferably an agar based culture medium.

20. Method according to claim 18 or claim 19, wherein the second cell culture is grown and/or maintained in a substantially liquid cell culture medium.

21. Method according to any one of claims 18-20 comprising, after co- culturing of the cells, determining a composition of the first and/or second culture medium.

22. Method according to claim 21 comprising comparing the composition of the first and/or second culture medium with a reference composition.

23. Method according to claim 22 comprising identifying an eliciting compound and/or an growth-modulating compound in the composition of the first and/or second culture medium.

24. Method according to claim 23 comprising isolating the eliciting compound and/or the growth- modulating compound from the culture medium.

25. Method according to claim 24 comprising assessing a growth- modulating compound for its ability to promote growth of a reference cell line.

26. Method according to claim 24 or 25 comprising assessing a growth-modulating compound for its ability to inhibit growth of a reference cell line such as a cancer cell line.

27. Method according to any of claims claim 24-26 comprising assessing an elicitor compound for its ability to activate an expression of silent bio-synthetic gene clusters in a reference cell line that encode for a growth-modulating compound.