NL2025992B1 - Multiwell plate for 3D cell culturing and imaging, method for manufacturing and uses thereof. - Google Patents

Abstract

Disclosed are devices and methods for cell culturing and cell analysis, more in particular to multi-well devices designed for growth, maintenance and/or microscopic analysis of cell culture models, including 3D cell culture models such as cell aggregates or spheroids. The disclosed device comprises a plurality of wells for propagating and imaging 3D cell cultures, the device being a unitary, optically transparent, structure including a plurality of wells, wherein the bottom of each well comprises a recessed imaging section and a closed bottom end, which closed bottom end is formed by an optically clear and flat bottom layer having a thickness of less than about 250 um, which bottom layer is integrally formed with the side wall of the bottom section and which interconnects the closed bottom ends of each of said plurality of wells to configure a device having a clear and flat bottom surface.

Description

P120276NL00 Title: Multiwell plate for 3D cell culturing and imaging, method for manufacturing and uses thereof.

The present disclosure relates to devices and methods for cell culturing and cell analysis. More in particular, it relates to multi-well devices designed for growth, maintenance and/or microscopic analysis of cell culture models, preferably 3D cell culture models, like cell aggregates or spheroids. It also relates to methods for manufacturing the multi-well device, and methods for spheroid handling using the device.

In vitro cellular and tissue models for various drug testing and screening experiments are often central to the development of novel therapeutics in the pharmaceutical industry. However, most in vitro studies are currently still performed under conventional two-dimensional (2D) cell culture systems, which are often not physiological models and/or hard to relate to functional tissues and tumors. Therefore, drug studies involving such models may not produce sufficiently accurate or realistic results. To obtain more meaningful results, in vivo studies involving animals are often utilized. However, major drawbacks of in vivo studies include the ethical aspects of using animal models and the time-consuming and expensive nature of these experiments.

In order to bridge the gap between the non-physiological conventional 2D models and in vivo experiments, 3D in vitro cell models have been developed in the art to provide more therapeutically predictive and physiologically relevant results, e.g. for drug testing and screening in the pharmaceutical industry.

One way to create 3D cell culture models is through the formation of so-called spheroids, or 3D clusters or aggregates of multiple cells or more complex and functional organoids.

A major purpose of growing 3D cell culture models in vitro, such as 3D cell spheroids, is to test pharmacokinetic and pharmacodynamic effects of drugs in preclinical trials. Toxicology studies known in the art have shown 3D cell cultures to be nearly on par with in vivo studies for the purposes of testing toxicity of drug compounds. For example, when comparing LD50 values for 6 common drugs, the 3D spheroid values were found to correlate directly with those from in vivo studies. Although 2D cell cultures were previously used to test for toxicity along with zn vivo studies, the 3D spheroids are better at testing chronic exposure toxicity because of their longer life spans. Thus, a 3D arrangement allows the cell cultures to provide a model that more accurately resembles (human) tissue in vivo without the need of using of verification in animal test subjects.

However, it has been observed in practice that the scaling up of 3D cell cultures in a manner that allows for certain industrial applications, such as high-throughput drug screening and testing, has several drawbacks. For example, one way of traditional spheroid formation involves cultivation of suspended cells in hanging drops on the underside of a Petri dish lid. This process requires inverting of the lid following placement of the drops. As a result, the drops are susceptible to perturbation, resulting in falling, spreading, and merging with neighboring drops. Although inexpensive, this method is labor-intensive, does not permit efficient scalable production, and is not compatible with automated instruments for high-throughput screening. Because 1t 1s difficult to perform media exchange without damaging the spheroids, this method usually requires another labor- intensive step of transferring the spheroids manually, one by one, to a multi-well culture plate for longer-term culture, treatment, analysis, and harvest.

In the instance of spheroids, an alternative is to induce the formation of spheroids under continuous agitation of a cell suspension in bioreactors, such as spinner flasks and rotary culture vessels. This method requires the consumption of large quantities of culture media. It also requires specialized equipment and the size and uniformity of the spheroids are hard to control. The high variability in spheroids prohibits their use in many applications.

Methods are also available to produce spheroids using 3D microwell structures and planar micropatterns. Microplates allow for a uniform, single spheroid formation across all wells and the culturing and assaying of spheroids in the same microplate, without the need for transfer to a new plate. A special (e.g. Ultra-Low Attachment; ULA) coating can be applied on the well surface to avoid cellular adhesion and promote cellular aggregation by gravitational force. The microplate format also enables media exchange or spheroid treatment with test compounds, drugs or assay reagent. For example, US2014/0322806 discloses a spheroid cell culture article including a frame having a chamber including an opaque side wall surface, a top aperture, a gas-permeable, transparent bottom, and optionally a chamber annex surface and second bottom, and wherein at least a portion of the transparent bottom includes at least one concave arcuate surface.

3D cell culture analysis by automated visualization plays an important role in drug testing and screening applications. For instance, majority of high-throughput spheroid assays involve microscopic detection of viable or dead cells, (morphological) changes of cells or cell organelles. Most automated imagers for analyzing standardized microplates are designed for “reading” the well through an optically clear ultra-flat bottom plate.

Typically, this reading uses high numerical aperture (NA) water-immersion objectives and requires all well access.

The microplate standards govern various characteristics of a microplate including well dimensions (e.g. diameter, spacing and depth) as well as plate properties (e.g. dimensions and rigidity), which allows interoperability between microplates, instrumentation and equipment from different suppliers, and is particularly important in laboratory automation. In 2010, the Society for Biomolecular Sciences (SBS) merged with the Association for Laboratory Automation (ALA) to form a new organisation, the Society for Laboratory Automation and Screening (SLAS). Henceforth, the microplate standards are also known as ANSI/SLAS standards. Currently known (standardized) microplates designed for 3D cell culture handling suffer from the drawback that their application for automated visualization is not optimal. More specifically, the arcuate surface of round bottom well plates designed to promote the formation of spheroids and other types of 3D cell cultures (e.g. each well bottom of a plate according to US2014/0322806) typically significantly limits their compatibility with commonly used automated plate imagers, in particular those that rely on imaging with water immersion lenses like the Operetta CLS imaging system and similar high content imaging systems.

This issue was addressed in WO2017/142410 in the name of the applicant, disclosing a round-bottom, optically clear insert plate to seed and grow spheroids, which insert plate can be combined to fit into a flat microplate designed for analysis using conventional high content imaging systems. The optical imaging is performed by focusing through the flat, clear-bottom of the microplate on the spheroids contained in the wells of the insert plate. This approach provides a convenient and cost-effective spheroid culturing and imaging system. However, for plates containing a higher number of wells, e.g. having a 384-well format, it was found technically challenging to precisely align the well bottoms of the insert plate with those of the microplate such that the accuracy of automated microscopic imaging is ensured.

Therefore, the inventors set out to develop a new 3D cell culture plate concept that allows not only for convenient 3D cell culturing and handling, but also for automated microscopic 3D cell culture analysis by commonly used automated plate imagers of plates comprising a high number (e.g. more than 96) of wells.

It was surprisingly found that this can be achieved by the provision of an injected molded unitary device of an optically transparent 5 material, the device comprising a plurality of tapered wells, wherein the bottom of each well comprises a recessed imaging section having a closed bottom end formed by an optically clear and flat bottom layer having a thickness of less than 250 pm. In a device as disclosed, the flat bottom layer is integrally formed with the side walls of the tapered wells and interconnects the closed bottom ends of each well to configure a device having an optically clear and flat (i.e. continuous and uninterrupted) bottom surface. This allows for improved viewing, at or from the well bottom, of the contents of the recessed imaging section using automated optical imaging systems, including those relying on water immersion lenses.

Accordingly, the present disclosure includes a device for use in propagating and imaging 3D cell cultures , the device comprising a plurality of wells, the device being a unitary, optically transparent, structure including a plurality of (tapered) wells, wherein the bottom of each well comprises a recessed imaging section having a closed bottom end formed by an optically clear and flat bottom layer having a thickness of less than approximately 250 um, which bottom layer is integrally formed with the side wall of the bottom section and which interconnects the closed bottom ends of each of said plurality of wells to configure a device having a clear and flat (i.e. uninterrupted) bottom surface.

This is fundamentally different from, for example, the multiwell plates marketed by InSphero AG (Schlieren, CH) under the tradename Akura™ — 96 microplate, representing an automation-compatible platform for the generation, long-term cultivation, observation and testing of scaffold- free 3D microtissue spheroids in 96-well format. Whereas these plastic plates are suitable for use in automated imaging equipment, such as the

SCREEN Cell3iMager and PerkinElmer Operetta CLS imaging systems, the clear imaging bottoms are not interconnected as a flat, single bottom layer. This hampers analysis using water immersion lenses, wherein the lens should ideally remain in fluid contact with the bottom of the plate when switching between the imaging of adjacent wells.

Also known is the GravityTRAP™ 384 or Akura™ 384 platforms which include distinct microtissue culture and media chambers separated by a tapered pipetting ledge, providing safe medium aspiration and exchange without accidental loss of microtissues; and a continuous flat- bottomed glass base plate combined with a black-walled body for confocal and high content imaging optics while reducing fluorescent cross-talk between wells. However, the separate manufacture and subsequent assembly of the plastic base plate comprising the wells and the glass base plate is time labor intensive and economically unattractive. In addition, gluing of the glass plate to the base plate bearing the narrow apertures can carry the risk of toxic glue components or residues entering into the well, which can hamper proliferation of cells.

In one embodiment, the disclosed multiwell-device is a unitary, optically transparent, structure including a plurality of wells, wherein each well comprises (1) a top section with an open upper end and an open lower end; (11) a middle section with an open upper end and an open lower end and (111) a bottom imaging section with an open upper end and a closed bottom end, wherein the top section, middle section and bottom imaging section are in fluid communication, and wherein the sidewall of the middle section converges towards the bottom section, wherein the closed bottom end is formed by an optically clear and flat bottom layer having a thickness of less than approximately 250 um, which bottom layer is integrally formed with the side wall of the bottom section and which interconnects the closed bottom ends of each of said bottom imaging sections to form a device having aclear and flat bottom surface.

In certain embodiments, the bottom surface of the device has a thickness of up to approximately 150 um, while in other embodiments the thickness may only be up to approximately 100 um. This is advantageously accompanied by an inner well flatness of less than or equal to approximately 20 um.

An individual well of a device of the present disclosure can generally contain a volume of fluid of between about 100 nanoliter and about 150 ul. Typically, the dimensions (e.g. volume) of each of the plurality of wells is approximately the same. In an example, a well is configured to contain about 1 pL to about 100 pL , in some embodiments the wells are configured to contain about 3 pL to about 80 pL, and in some embodiments the wells are configured to contain about 5 pL to about 70 nL. In embodiments, the well volume can be less than about 60 ul. The specific design (e.g. height, cross-section, diameters, angle, and the like) of the well can vary according to needs. In many instances, wells have a circular or square opening on the surface of the microplate, however, a microplate herein can have wells with a surface area with any shape. A well can be a space having a width, a depth, and an opening surrounded by a sidewall and a bottom surface.

The bottom of the well can be planar, concave or convex. The bottom of the well is opposed to the opening of the reaction chamber In one embodiment, the total height (depth) of a well is in a range of about 8 mm to about 12 mm, and in other embodiments less than about 10 mm, while in yet other embodiments, in a range of about 8.2 mm to about

9.5 mm. In a specific aspect, the disclosure comprises a unitary multiwell device having one or more of the following features: (1) the cross-section of said closed bottom end is in a range of about 0.2 mm? to about 0.8 mm:2; (ij) the inner well flatness is less than or equal to about 20 um; (iii) the volume of each well is less than about 80 ul, and in some embodiments, less than about 70 ul; (iv) the total height of each well is in a range of about 8 mm to about 12 mm, and in some embodiments, in a range about 8 mm to about 10 mm; and, (v) a bottom off-set of less than about 300 pm, and in some embodiments, less than about 250 um, while in other embodiments, less than about 200 pm.

The expression “bottom imaging section” and the “recessed imaging section” , or merely “imaging section” are herein used interchangeably.

The opening of a well can be any shape for example substantially round, square, oval, rectangular, hexagonal, crescent, or star-shaped. The different sections of the well (top section, middle section and bottom imaging section) can be any shape, for example, substantially round, square, oval, rectangular, hexagonal, crescent, or star-shaped. In one embodiment, the cross-section of each section of the well is circular.

In one embodiment, the length of the inner edge or inner diameter of the open end of the top section is at least about 3.0 mm, and may be at least about 3.1 mm, or at least about 3.2 mm. The length of the inner edge or inner diameter of the open end of the top section imay be less than about

3.6 mm.

In one embodiment, the height ratio between the top section and the middle section of a well is in a range of from about 20:1 to about 2:1, and in some embodiments a range of about 10:1 to about 4:1, and in yet other embodiments a range of about 8:1 to about 5:1. In one aspect, the height of the top section 1s about 5 mm to about 10 mm, and in some embodiments may be about 6 mm to about 10 mm, and/or a height of the middle section is about 0.5 mm to about 2.0 mm, while in other embodiments it 1s about 1.0 mm to about 1.7 mm.

The recessed imaging section may be concentrically arranged with respect to the longitudinal axis of at least one remainder portion of the same well. For example, the imaging section may be concentrically arranged with respect to the longitudinal axis of the middle section of the same well, optionally wherein the middle section is concentrically arranged with respect to the longitudinal axis of the top section of the same well.

Typically, the volume of the imaging section of each of the plurality of wells is the same, and has a volume up to about 0.35 ul. In some embodiments, the volume is up to about 0.30 ul, for example, in a range of about about 0.05 ul to about 0.25 ul. In a specific aspect, the volume isin a range of about 0.08 ul to about 0.20 ul. The desired volume can be obtained using different designs (height, cross-section). In one aspect, the bottom imaging section has a height in a range of about 0.20 mm to about 0.65 mm, including a range of about 0.30 mm to about 0.50 mm. To allow for 3D cell formation and imaging, including but not limited to spheroid formation an imaging, it was found that a cross-section of the closed bottom end of the imaging section may be in a range of about 0.2 mm? to about 1.0 mm?2. The side walls of said bottom imaging section may be essentially perpendicular with respect to the flat bottom surface of the device.

To ensure that the multiple cells that are introduced into the well can effectively propagate and form 3D clusters or aggregates, it is desirable that the cells become concentrated in the bottom imaging section. To that end, the side walls of the middle section of the well converge towards the bottom section. In some embodiments, the side walls of the middle section have an angle in a range of about 35° to about50° with respect to the flat bottom surface of the device.

To facilitate its use in automated handling and/or imaging systems, the disclosed device may have a footprint of a standard SBS/ANSI format multiwell plate. For example, a dimension of the multiwell plate device, a well dimension, and/or a well spacing may conform to the SBS (Society for Biomolecular Screening) standard, also known in the art as

ANSI/SLAS Microplate Standards. The dimensions for typical multiwell plates conforming to the SBS standard are 127.76 mm + 0.25 mm (length) and 85.48 mm + 0.25 mm (width) with the wells arranged in a standardized format depending on the total number of wells. This ensures that the multiwell plate device is compatible for use in workstations or automated sample preparation systems specifically adapted to handle SBS conforming multiwell plates.

The design of the wells can vary. In one embodiment, the multi- well plate is a 96- well or 384-well SBS-standardized plate.

For example, the wells can have a circular or square cross section. In a specific aspect, the plate is a 384-well SBS-standardized microplate containing 384 wells having a circular cross section. In another embodiment, the microplate 1s a 96-well SBS-standardized microplate containing 96 wells having a square cross section.

In order to prevent or reduce collision of objectives against the microplate rim, the disclosed devices may have a bottom off-set of less than about300 um, however, it may be less than about 250 pm, or in other instances, less than about 200 um. This allows the use of each well of the device, including those positioned at the edges.

The disclosed devices may comprise a row of wells or an array of wells. For example, it can have 2, 6, 24, 48, 96, 384 or more wells. The size and shape of the devices can vary greatly as is known in the art. The layout of the wells can also vary greatly, for example, many known multiwell microplates have a plurality of wells arranged in a 2:3 rectangular matrix. Other microplates have a plurality of wells arrangedin a 1: 1 or 3 :4 matrix. A device as described herein can have any layout of wells as would be obvious to one skilled in the art.

In one embodiment, a device includes an array of 384 wells arranged in 16 by 24 array, for example, with a center-to-center well spacing of 4.5 mm.

In another embodiment, a device has a footprint of a standard microscope slide, for example, wherein the device measures about 75 mm by about 25 mm.

For example, the disclosure provides a device comprising a plurality of wells for use in propagating and optical analysis by a manual microscope of cell spheroids, the device being a unitary, optically transparent structure having the footprint of a standard microscope slide and comprising up to about 50, including up to about 40, such as about any of 36, 30, 25, 20, 18, 16, 12, 9, 8 wells, which may be arranged in a row or array of wells, e.g. 3x3, 3x4, 4x4, 5x5 wells.

A unitary multi-well device of the present disclosure is among others characterized in that the closed bottom ends of the imaging sections are formed by an optically clear and flat bottom layer which is integrally formed with the side wall of the bottom section and which interconnects the closed bottom ends of each of said plurality of wells to configure a device having a clear and flat (i.e. uninterrupted) bottom surface having a thickness of less than about 250 um.

This is especially important when optical imaging is performed using an objective with a high numerical aperture (NA). The NA is commonly used in microscopy to describe the acceptance cone of an objective and hence its light-gathering ability and resolution.

High NA (hNA) objectives have shorter exposure times and thinner depth-of-focus, and are typically made for analysis through a thin (typically up to about 180 um) bottom in order to allow for high resolution optical imaging of spheroids comprised in the imaging section.

The multi-well device may further comprise a seal, lid or cover plate that is positioned over said plurality of wells, for instance to protect the cells and/or spheroids during culturing, incubating, handling and/or imaging.

It was surprisingly found that a device having an integral bottom layer of this thickness and flatness can be produced as a unitary piece by injection moulding. Accordingly, the present disclosure also provides a method for providing a device as described herein above, said method comprising the manufacture of the disclosed devices as one piece by injection moulding of a molten transparent polymer in an injection moulding machine. In one embodiment, a method according to the present disclosure comprises only a single cycle of molten polymer injection.

The process of injection moulding is one of the most common and recognizable steps in the plastic manufacturing process. Injection moulding is valuable due to its low labour costs, minimal wastage and high production rates. Typically, the injection molding process comprises the following steps. In step 1, the injection mould closes, clamping together the two halves of the injection mould, and the cycle timer begins. Then, a heated plastic (polymer) composition is injected into the mould while the displaced air escapes along the parting line and along the ejector pins or injection nozzle. The runner, vent design and gate ensure that the mould is filled correctly. Once the mould has been filled, the part cools down. The piece is given the right amount of cool-down time needed to harden the material used. During the cool-down period, a barrel screw retracts and draws new plastic resin into the barrel. This plastic resin is drawn in from the material hopper. Heater bands maintain the necessary barrel temperature for the type of resin used. Finally, the mould opens while the ejector rod shifts the ejector pins forward. This step causes the part to fall into a bin located under the mould.

The unused sprues and runners can be recycled to be used in future moulds.

A method according to the present disclosure can be performed using a standard system for injection molding known in the art. Typically, injection molding systems comprise a mold placed in between a clamping mechanism. The clamping mechanism can move towards the final produced device. An injection cylinder comprising an injection screw is connected to the mold. The temperature of the cylinder is maintained by cylinder heaters that are placed around the cylinder. A motor is placed at the start of the injection molding apparatus. It is connected to the screw and cylinder in a known manner to operate the system. Molten transparent polymer material is introduced into the mold via an injection nozzle. By moving the mold sides apart and ejecting the produced part using ejectors, a final product will be released.

The transparent polymer preferably has a melt flow index (MFI) in the range of about 20 to about 70. For example, the MFI is about 20 to about 40, or about 30 to about 70, or about 40 to about 60. In one embodiment, the transparent polymer has a refractive Index (nD; 589 nm) in a range of about

1.52 to about 1.57.

Very good results were obtained by injection moulding a polymer comprising cyclin olefin co-polymer (COC), cyclic olefin polymer (COP) or polystyrene (PS). Hence, the present disclosure includes a device that is made from COC, COP or PS, either as a single material or as the major material representing for example at least 80w%, preferably at least 90wt%, more preferably at least 95wt% of the polymer used.

In some embodiments, for example for 3D culturing applications, at least the bottom imaging section of at least one well is provided with an ultra- low (mammalian) cell attachment coating. Such coatings are well known in the art.

The present disclosure also provides a method of 3D cell culture handling, COMPYISING: a) mserting a plurality of cells into at least one well of a unitary multiwell device as disclosed herein, and allowing the formation of 3D cell culture s; and; b) performing on one or more of the 3D cell cultures a handling method that comprises 3D cell culture culturing, 3D cell culture maintaining, 3D cell culture analysis, 3D cell culture testing, and combinations thereof.

As used herein, the term “3D cell culture” encompasses spheroids, 3D clusters or aggregates of multiple cells as well as more complex and functional organoids or organoid-like structures. In a specific aspect, the present disclosure provides a method for spheroid handling, comprising a) mserting a plurality of cells into at least one well of a unitary multiwell device according to the disclosure, and allowing the formation of spheroids; and: b) performing on one ox more of the spheroid handling methods comprising spheroid culturing, spheroid maintaining, spheroid analysis, spheroid testing, and combinations thereof The formation of 3D cell cultures such as spheroids can be achieved by methods known in the art, typically involving gravity and/or cellular interactions. Depending on the type of cells, one or more agents can be added to the growth medium to promote 3D cell culture formation or mamtenance. For example, it has been shown in the art that growth factors support the clustering and/or proliferation of cells, In embodiments, a 3D cell culture handling method according to the present disclosure comprises optical imaging of 3D cell cultures, including using an automated microplate imager. In a specific aspect, optical imaging involves high resclution fluorescence and/or brightfield microscopy in a high- throughput manner with a High Content Screening system.

A method of 3D cell culture handling as provided herein may comprise contacting the 3D cell culture with a potential therapeutic agent. In one embodiment, the potential therapeutic agent is at least one of a. a molecule comprising an endogenous ligand or ligands, a biological sample suspected of containing a native or endogenous ligand or hgands, a combinatorial library of small molecules, a hormone, an antibody, a polysaccharide, an anti-cancer agent, a natural product, a terrestrial product and a marine natural product; b. a molecule that binds with high affinity to a lnopolymer , the molecule comprsing a protein, a nucleic acid, and a polysaccharide; and, c. a purified biological molecule comprising a protein, a nucleic acid, a silencing RNA (siRNA), a micro RNA (miRNA), and a short hairpin RNA (shRNA).

In some embodiments, the 3D cell culture handling method further comprises assaying for a marker indicative of modulation of a cellular target of said potential therapeutic agent or screening for activity in modulating the phenotype of a 3D cell culture, In one embodiment, the cells forming the 3D cell culture, are transformed with at least one heterologous nucleic acid molecule that encodes one or more biomarkers associated with a phenotype of interest. In an embodiment, the recombinant nucleic acid molecules} are chromosomally integrated into the genome of the cell.

In embodiments, the biomarkers are linked to an indicator that can be detected in situ following expression of the biomarker. The term “indicator” is meant to refer to a chemical species or compound that 1s readily detectable using a standard detection technique, such as dark versus light detection, fluorescence or {chemiluminescence spectrophotometry, scintillation spectroscopy, chromatography, liquid chromatography/mass spectroscopy (LOMS), colorimetry, and the like. Representative indicator compounds thus include, but are not limited to, fluorogenic or fluorescent compounds, chemiluminescent compounds, calorimetric compounds, UV/VIS absorbing compounds, radionucleotides and combinations thereof. Exemplary indicators for use in the screening methods of the present disclosure are proteins including Red Fluorescence Protein (RFP), which fluoresces when exposed to light of wavelength 558 nm, Green Fluorescent Protein (GFP) which fluoresces when exposed to light of wavelength 395 nm, and Juciferase, which produces light in the conversion of luciferin and oxygen to oxyluciferin. These indicators, when coupled with an automated plate reading mechanism, form an embodiment of the present disclosure that is readily amenable to both robotic and very high throughput systems.

Whereas any cell type of interest can be used, in many embodiments they are capable of sustained growth under tissue culture conditions. For example, the cells are neoplastic cells. Cells may be tumor cells that have been isolated from a human. In a specific embodiment, the cells are human malignant tumor cells comprising breast cancer, lung cancer, prostate cancer, colon cancer, melanoma cancer, and cancer of the bone and connective tissues, In another specific aspect, the cells are stem cells comprising embryonic stem cells or adult stem cells, progenitor cells, bone marrow stromal cells macrophages, fibroblast cells, endothelial cells, epithelial cells, and mesenchymal cells. The disclosure alse includes the use of a multi-well device or 3D cell culture handling method as described herein in a high throughput drug discovery program. For example, the disclosure provides an industrially applicable high throughput screening method for assaying the non-, pro-, or anti- apoptotic or proliferative or necrotic activity of test compounds in 3D cell culture, like spheroid cells. Compounds discovered using the screening methodologies of this disclosure can be developed into therapeutics that systemically target the inhibition of metastasis. Additionally, these techniques can be adapted to conduct individualized treatment on patients in the clinical setting. For example, primary breast carcinoma cells isolated from patients may be cultured in the 3D cell culture screening systems of the present disclosure and screened against known lead compounds from previous chemical hbrary screenings as well as clinical agents in use today in order to identity patients that are hkely or unhkely to respond to one or more agents available to the treating physician ("personalized medicine/therapy™).

LEGEND TO THE FIGURES Figure 1: Cross sectional perspective view of an embodiment of a device according to the present disclosure. The unitary multi-well plate (1) comprises a plurality of wells (2). The bottom section of each well (2) comprises a recessed imaging section (3) and a closed bottom end (4) formed by an optically clear and flat bottom layer (5) having a thickness of less than about 250 pm. The bottom layer (5) is integrally formed with the side wall (6) of the bottom section and which interconnects the closed bottom ends of each of said plurality of wells to configure a device having a flat and uninterrupted bottom surface (10).

Figure 2: Embodiment of a device according to the disclosure, configured as multi-well plate (1), shown in a cross sectional view. The multi-well plate (1) comprises a plurality of wells (2) arranged in an array, each well (2) comprising a bottom imaging section (3).

Figure 3: Enlarged cross sectional view of a portion of the device (1) shown in Figure 2. Each well (2) comprises (i) a top section (7) with an open upper end (7a) and an open lower end (7b); (11) a middle section (8) with an open upper end (8a) and an open lower end (8b) and (111) a bottom imaging section (9) with an open upper end (9a) and a closed bottom end (9b), wherein the top section (7), middle section (8) and bottom imaging section (9) are in fluid communication, and wherein the sidewall (8c) of the middle section converges towards the bottom section (9).

Figure 4: is a top view of the multi-well plate (1) shown in Figure 2 comprising a plurality of wells (2) having a circular cross-sectional area at the open upper end (7a) of the top section. Each well contains a recessed imaging section (9) having a circular cross-sectional area, the recessed imaging section arranged concentrically with respect to the longitudinal axis of the remainder portion of the same well. In the embodiment shown, the array includes 354 wells arranged in 18 rows and 24 columns. The multi-well plate comprises an orientation notch (11) in the lower left corner when the plate is oriented with well Al in the upper left hand corner.

Figure 5: Time course of HepG2 spheroid formation in a unitary multi-well device according to the disclosure. Development of spheroids is visible in recessed imaging section. Panel A: Day 0, t= 0.1h; Panel B: Day 0, t = 4h; Panel C: Day 5.

Figure 6: Fluorescent imaging of 5 day old HepG2 spheroids in a unitary multi-well device according to the disclosure (20x hNA objective, confocal mode). Panel A: bright field image, 100 pm height; Panel B: DNA counterstain, 25 um height; Panel C: live and dead cell stain, 25 um height.

EXPERIMENTAL SECTION Example 1: Manufacture of a multi-well device This example exemplifies the manufacture of a unitary (i.e. single piece) multi-well device according to the disclosure using established injection moulding technology, see for example US7959844B1. For producing a 4x4 multi-well microslide device an Arburg 50T injection molding machine was used. The hopper feeder was set at a temperature of 40°C. The five separate cylinder heaters were set at temperatures ranging from 240 to 265 °C. The back and front side of the mold were maintained at a temperature of 90°C. The injection screw operated at a pressure of 80 bar, at 10 rpm, with a dosing time of 1.1 second in order to accumulate the melted resin needed for injection into the mold. Melted cyclic olefin polymer (COP) resin was injected into the mold at an injection pressure of 950 bar, with an injection speed of 40cm3/sec for 0.40 second. A second pressure step or holding pressure was performed at 325 bar, with a flow of resin of 15 cm3/sec for 2.1 sec to remove residual air and to compensate for shrinkage of the material after cooling down of the injected resin. The total injection molding cycle time, including opening of the system and ejection of the device, for producing a single multi-well device was 30 seconds. 3.35 grams of material was used to produce the device, of which

2.61 grams was required for the final multi-well device and 0.74 grams of solidified left over material that came out of the injection nozzle.

Example 2: Formation and propagation of spheroids This example describes spheroid generation of two mammalian cell lines in a multiwell device according to Example 1. The wells were coated with an ultra low attachment (ULA’) coating solution as described previously in WO2017/142410. HepG2 (ATCC HB-8065, LGC Standards GmbH, Wesel, Germany) and HeLa (ATCC CCL-2, LGC Standards GmbH, Wesel, Germany) cells were maintained in T25 cell culture flasks (734-2064, Nunc, Roskilde, Denmark) using DMEM/F12 + Glutamax culture medium (HepG2, 31331-028, Life Technologies) or RPMI-1640 (HeLa, 61870-044, Life Technologies), supplemented with 10% Fetal Bovine Serum (10500-064, Life Technologies) and 1% Penicillin-Streptomycin (15070-063, Life Technologies).

Cells were detached by trypsinization (8.5% trypsin-EDTA solution in PBS (15400-054, Life Technologies} at approximately 80% confluency. A single cell solution was prepared by dispersing the cell suspension at least ten times at the wall of the flask. Cell concentration was determined using a Coulter Particle Counter (Beckman Coulter, Woerden, The Netherlands). Five hundred cells in a total volume of 30 ul were seeded into each well of the ULA coated multi-well device and spun down (500 rpm, for 2 minutes) to the bottom of the well. The device was sealed with a breathable sealing membrane (Breathe-Easy, Z380059, Sigma-Aldrich) to prevent evaporation and contamination of the culture medium. Cells were grown in an incubator at 37°C and 5% CO: and spheroid formation was followed over time by visual microscopic inspection through the clear and uninterrupted flat bottom surface of the device using aHund Wetzlar Wilovert S inverted light microscope (Helmut Hund Gmbh, Wetzlar, Germany). Typically, spheroid formation in the bottomed recessed imaging sections of the wells was observed to be complete after 3 to 5 days.

See Figure 5 and 6 for exemplary images.

Example 3: Microscopic imaging of spheroids

This example demonstrates the advantageous properties of a unitary device according to the disclosure (see Example 1) for the microscopic imaging of spheroids in comparison with three prior art multi-well devices (Corning Spheroid 384-well plate; Akura™ 96 and CellCarrier Spheroid-96 well plate). Cells (Hela (Corning plate); HepG2 (Akura-96; CellCarrier Spheroid ULA- 96-well; 4x4 microslide device) were seeded and propagated as described in Example 2. After 3 days, the wells were inspected microscopically as described in Example 2 and tested for Long Working Distance (LWD) and high NA objective compatibility.

Results are shown in the table below.

Table 1 : Microscopic imaging performance Bottom | Bottom Bottom 10x LWD 20x high NA type thickness | height objective, objective, NA =0.3 NA =0.75 Corning Round 90 um 1.75 mm | Possible Possible Spheroid bottom (56 of 384 (24 of 384 wells 384-well wells not not focused) plate focused) Akura™ 96 Possible Not possible | CellCarrier | Round 1.0 mm 3.1 mm Possible Not possible Spheroid-96 | hottom well Unitary Flat <190 um 0.2 mm Possible Possible microslide device

It can be concluded that a multi-well device according to the present disclosure shows improved spheroid imaging properties, in particular for high resolution imaging using high NA objectives.

Claims (24)

Conclusions A device comprising a plurality of wells for use in amplifying and imaging 3D cell cultures, including spheroids, the device being a unitary, optically transparent structure comprising multiple wells, the bottom of each well having a recessed imaging section and a closed bottom end, the closed bottom end being formed by an optically clear and flat bottom layer having a thickness of less than 250 µm, the bottom layer being integrally formed with the sidewall of the bottom end and comprising the closed bottom ends of each of the plurality of wells having connect to configure a device with a clear and flat bottom surface. A device according to claim 1, wherein the flat bottom layer has a thickness of up to about 150 µm, preferably up to about 100 µm. The device of claim 1 or 2, wherein each well (i) has a top portion having an open top end and an open bottom end; (ii) a center portion having an open top end and an open bottom end and (iil) a bottom imaging portion having an open top end and a closed bottom end, wherein the top portion, center portion and imaging portion are in open communication, and wherein the sidewall of the center portion converges to the lower part. The device of any one of claims 1 to 3, wherein the volume of the lower imaging section is in the range of about 0.05 to 0.25 µl. A device according to any one of the preceding claims, characterized in that it has one or more of the following features: (1) the cross section of the closed lower end is in the range of approximately 0.2 to 0.8 mm ?2; (ii) an inner well flatness is less than or equal to about 20 µm; (iii) the volume of each well is less than about 80 µl, preferably less than about 70 µl; (iv) the total height of each well is in the range of about 8 to 12 mm, preferably about 8 to about 10 mm; (v) a bottom off-set of less than about 300 µm, preferably less than about 250 µm, more preferably about 200 µm. The device of any preceding claim, wherein the device has a footprint of a standard SBS/ANSI format multiwell plate, preferably wherein the device is configured as a 384-well array. A device according to any one of the preceding claims, manufactured as a unitary construction by injection moulding. A device according to any one of the preceding claims, which is made of cyclic olefin copolymer (COC), cyclic olefin polymer (COP) or polystyrene. The apparatus of any preceding claim, wherein at least the recessed imaging section of the plurality of wells is provided with an ultra-low (mammalian) cell attachment coating. A method of providing a device according to any one of the preceding claims, wherein the method comprises manufacturing the device as one piece by injection molding a molten transparent polymer in an injection molding machine. The method of claim 10 comprising only a single cycle of molten polymer injection. The method of claim 10 or 11, wherein the transparent polymer has a melt flow index (MFT) in the range of about 20 to 70. The method of any one of claims 10 or 11, wherein the transparent polymer has a refractive index (nD; 589 nm) in the range of about 1.52 to about 1.57. The method of any one of claims 10 to 13, wherein the polymer comprises a cyclic olefin copolymer (COC), cyclic olefin polymer (COP) or polystyrene (PS). A method for handling 3D cell culture, preferably handling spheroids, comprising: a) introducing multiple cells into at least one well of the device according to any one of claims 1-9 and allowing the formation of a 3D cell culture; and b) performing one or more of the methods for handling 3D cell culture selected from the group consisting of 3D cell culture culture, 3D cell culture preservation, 3D cell culture analysis, 3D cell culture assays, and combinations thereof. The method of claim 15, wherein the method further comprises optical imaging. The method of claim 16, wherein performing optical imaging comprises high-resolution, high-throughput, high-resolution fluorescence and/or bright-field microscopy with a High Content Screening system. The method of any one of claims 15-17, further comprising contacting the 3D cell culture with a potential therapeutic agent. The method of claim 18, further comprising testing for a marker indicative of modulation of a cellular target of the potential therapeutic agent or screening for activity in modulating the phenotype of a 3D cell culture. A method according to any one of claims 15-19, wherein the method comprises handling spheroids. The method of any one of claims 15-20, wherein the cells are neoplastic cells. The method of any one of claims 15-20, wherein the plurality of cells are human malignant tumor cells selected from the group comprising at least one of breast cancer cells, lung cancer cells, prostate cancer cells, colon cancer cells, melanoma cancer cells, and cells related to cancer of the bone and connective tissue. The method of any one of claims 15-20, wherein the cells are stem cells comprising at least one of embryonic stem cells, adult stem cells, progenitor cells, bone marrow stromal cells, macrophages, fibroblast cells, endothelial cells, epithelial cells and mesenchymal cells. Use of a device according to any one of claims 1 to 9, or a method according to any one of claims 15 to 23, in a high-throughput drug discovery program.