AU2021377418A1 - Organoid culture platform - Google Patents

Abstract

There is a need to provide a system for culturing cells or cell clusters, such as organoids, whereby the location of the cell, or cell cluster, is fixed within the culture system. This permits easy localisation of an individual cell or cell cluster. Preferably, this is done in a manner that still allows for development of the cell or cell cluster in a biologically accurate way and can be readily used with high-throughput liquid handling techniques and analysis techniques, such as microscopy. This may be addressed by providing a cell culture insert, which provides a suspended cell growth platform which allows a cell, or cell cluster, to be cultured in an inverted position at a predetermined distance from the bottom of a cell culture plate.

Description

Title of Invention

ORGANOID CULTURE PLATFORM

Priority Claim

[0001] This application claims priority from Australian patent application 2020904171 filed on 13 November 2020, the entire content of this application is incorporated herein.

Technical Field

[0002] The present invention relates to cell culture inserts for use with multi-well plates for growing cells, cell clusters or cell aggregates in vitro. The invention further relates to methods and cell culture systems for growing cell or cell clusters. Specifically, cell culture inserts, systems and methods are provided which allow for high-throughput screening and visualisation of cells grown in three-dimensions.

Background of Invention

[0003] The ability to study biological systems in a tightly controlled and reproducible environment is a cornerstone of biological research. Typically, biological systems are studied in either an in vivo environment or within an in vitro environment. In vivo systems have the advantage of more accurately replicating the natural system which comprises many different interacting cells as well as variations such as nutrient and gas gradients. However, in vivo systems typically do not permit real time observation of cellular processes.

[0004] Further, in vivo systems require animal models which also presents numerous problems. Primarily, animal models are often poor proxies for the human states or diseases which are being studied. Therefore, the results generated within animal models may not necessarily be extrapolated to humans. Further, the use of animals in research is strictly regulated by ethics, which require that the number of animals be kept to a minimum. These issues and limitations, in combination with the cost of housing animals drives a need for alternative means for studying biological systems that do not rely on animals. [0005] Consequently, researchers often use in vitro cell culture to study cells in an animal free and controlled environment. While in vitro methods are cheaper, more humane, can use human cells and are often more easily replicated, they are often poor at replicating the natural environment in which the cultured cells exist. To overcome this limitation, researchers have been able to grow cells in three dimensional clusters using support scaffolds such as extracellular matrices. When grown in the presence of appropriately timed growth factors and in the appropriate manner, these cell clusters can differentiate into varying cell types that self-organise into structures that more accurately replicate a tissue or organ as it exists in vivo. Essentially, they can form small versions of different tissues. These types of differentiated cell aggregates are called organoids.

[0006] Methods are known in the art for generating a variety of organoids from non- terminally differentiated primary cells or cell lines. Currently, to generated organoids, individual cells are cultured in bulk in the presence of specifically timed growth factors and nutritional media.

[0007] An example of the generation of brain organoids is provided in Lancaster, M. and Knoblich, J., “Generation of cerebral organoids from human pluripotent stem cells”, Nature Protocols (2014), 9(10), 2329. In brief, human stem cells are cultured in bulk and aggregated to form cell clusters called embryoid bodies. Each individual embryoid body is transferred into a separate well of a Il-bottom multi-well plate, which is coated with non-adhesive compounds. The embryoid bodies are then cultured in varying media that induce the formation of different cell types, followed by transfer to a floating droplet of Matrigel™ to promote outgrowth of neuroepithelial buds. The tissues are then transferred, in bulk, to a spinning bioreactor or an orbital shaking plate to improve nutrient and gas exchange, and to allow more extensive growth and further development into defined brain regions.

[0008] While such a protocol allows for generation of brain organoids, it is difficult to study the development of each organoid throughout the stages of development, particularly in later development when the organoids are in a constant state of movement. Further, these bulk systems do not allow assessment of how interventions may alter the development of the organoid relative to each other as all of the organoids are typically incubated in the same medium and the location and orientation of each organoid is consistently changing. While this can be overcome by placing organoids in multiple spinning bioreactors or multiple orbital shaking plates, these are relatively large devices which take up considerable space, and the constant movement of the organoid prevents simple visualisation without having to manipulate the organoid. Furthermore, the orientation of the organoid is not consistent and therefore it is not possible to effectively analyse the development of specific features, or cells, within the organoid.

[0009] A similar method is disclosed in WO 2003/004626 A3. Here a stirred bulk culture is used to ensure the media conditions are the same throughout the culture at any given time. Again, this consistent agitation, and the culture of multiple organoids together, makes repeated assessment of individual organoids over time very difficult.

[0010] There is a need to provide a system for culturing cells and cell clusters, such as organoids, whereby the location of the cell, or cell cluster, is fixed within the culture system, and therefore permits easy localisation of an individual cell or cell cluster. Preferably, this is done in a manner that still allows for development of the cell or cell cluster in a biologically accurate way and can be readily used with high-throughput liquid handling techniques and analysis techniques, such as microscopy.

Summary of Invention

[0011] The present invention is predicated on the identification of the need to provide a means for high-throughput screening, particularly high-throughput imaging, of in vitro grown cells, cell clusters and organoids, which cannot be achieved by current cell culture systems.

[0012] Accordingly, the present invention provides a cell culture insert, for use with a multi-well plate, the insert including: at least one well insert adapted for insertion into a well of a multi-well plate; and at least one cell growth platform defining a lower surface, the at least one cell growth platform supported by the at least one well insert such that the lower surface of the at least one cell growth platform, when positioned within a well of a multi-well plate, is positioned at a predetermined distance from the bottom of a well.

[0013] The present invention, in use, provides an inverted cell growth platform that maintains a cell, or cell cluster, at a predetermined distance from the bottom of a multi- well culture plate. This allows the localisation of the cell, or cell cluster, in a set position relative to the cell culture plate and therefore allows for quick location of the cell, or cell cluster, in three-dimensional space relative to the multi-well plate. Further, in combination with a self-supporting scaffold, the cell culture insert restricts the orientation of the cell or cell cluster thereby allowing for the continued observation over time of features of the cell, or cell cluster, as well as maintaining the cell, or cell cluster, within the cell growth medium.

[0014] By providing a fixed location and orientation of the cell, or cell cluster, high- throughput screening can be performed on the cell culture. One particularly envisaged method of high-throughput screening is microscopic visualisation. Microscopic visualisation requires that the object for visualisation is within the working distance of the objective lens of the microscope. This objective working distance varies depending on the microscope and can be customised. However, in general the objective working distance is inversely related to the power of the microscope. Common objective working distances of standard microscopes are between 0.13mm to 4mm.

[0015] A cell, or cell cluster, growing on the cell culture insert of the present invention will be visualised from the bottom of a multi-well plate. Therefore, to allow for use of the cell culture insert of the present invention with common microscopes it is desirable to configure the cell culture insert such that the position of the lower surface of the at least one cell growth platform, in use, is at a predetermined distance and within the objective working distance of common microscopes. Such a microscope will be positioned below a well of a multi-well plate. Therefore, in some embodiments, the predetermined distance of the lower surface of the at least one cell growth platform from the bottom of the well is from 0.5mm to 4mm, or is from 0.5mm to 3.5mm, or is from 0.5mm to 3mm, or is from 1 mm to 2.5 mm, or is about 1 ,5mm, or is about 2mm or is about 2.5mm. Further, to enable effective visualisation, in some embodiments of the invention, the at least one cell growth platform is horizontal in use so that the objective working distance is maintained across the width of the cell growth platform.

[0016] Having confined the location of the cell, or cell cluster, in the Z axis (height) it is also desirable to limit the location of the cell, or cell cluster, in the horizontal X and Y axes. To allow for accurate localization of the cell, or cell cluster, within a well of a multi-well plate, particularly 24 and 48 well plates, it has been determined that the optimal width or diameter of the cell growth platform is from 2mm to 4mm, or from 2.5 to 3.5mm or is about 3mm. This allows for accurate localisation of the cell, or cell cluster, on the at least one cell growth platform (and hence within the well) and also allows for access by fluid handling devices needed to add, remove, or change cell culture media. Further, the at least one cell growth platform may include a peripheral annular rim which, in use, extends downward from the lower surface. This rim assists in the localization of the cell, or cell cluster, on the at least one cell growth platform making the cell, or sell cluster, easier to position by a user and making the location more predictable during visualisation.

[0017] To maintain the at least one cell growth platform at the predetermined distance, the cell culture insert may include a support for abutting and being supported by a multi-well plate, preferably with the at least one well insert attached to the support. Any support suitable for maintaining the at least one cell growth platform at the predetermined distance from the cell growth platform at the predetermined distance from the bottom of a well may be used with the present invention. For example, a series of legs may be provided which abut the bottom of a well in a multi-well plate thereby maintaining the at least one cell growth platform spaced-apart from the bottom of the well. However, in a preferred embodiment the support abuts and is supported by an upper surface of a multi-well plate. This allows the cell growth platform to be suspended within a well of a multi-well plate without touching the inside of the well. It also permits the support to be easily manipulated to insert or remove the at least one well insert and associated cell growth platform from a well of a multi-well plate.

[0018] To make manipulation of multiple well inserts easier, it may be advantageous to link the cell culture inserts together such that they can be manipulated at the same time. Therefore, in some embodiments, the support is attached to multiple well inserts, each insert with a corresponding cell growth platform. In this form, the cell growth insert has well inserts that are spaced apart and configured for use with specific multi-well plates such as 12, 24, 48 or 54 well plates. Accordingly, in some embodiments, the support includes 12, 24, 48 or 54 well inserts, each with a corresponding cell growth platform.

[0019] During the culturing of cells, it is typical to remove, add, or change cell culture media. Therefore, it is advantageous for the cell culture insert of the present invention to be configured to allow cell culture medium within a well to be aspirated without having to remove the cell culture insert. Consequently, in some embodiments, the support, the at least one well insert and the at least one growth platform are configured to provide an aperture to permit fluid to be added to a well and/or removed from a well when the insert is positioned in a multi-well plate. Preferably the aperture allows for fluid to be added and/or removed using a P10 to P1000 pipette tip, or a P100 to P1000 pipette tip. Alternatively, in some embodiments, including those which do not include a support, the at least one well insert and the at least one cell growth platform are configured to permit fluid to be added to a well and/or removed from a well when the insert is positioned in a multi-well plate.

[0020] Cell are typically grown in vitro within a cell culture medium which contains the required nutrients and conditions for cell growth. Therefore, it is advantageous to optimise the exposure of a cell or cell cluster to the medium. Therefore, in some embodiments of the invention, the lower surface of the at least one cell growth platform is configured to allow fluid communication around a cell, or cell cluster, positioned on the lower surface of the at least one cell growth platform. To facilitate this, in some embodiments, the at least one cell growth platform may include an upper surface and a lower surface which are in immediate fluid communication when the cell culture insert is in use with a well of a multi-well plate. The immediate fluid communication allows cell culture media added to the well to envelop the cell or cell cluster and allows for ready flow of nutrients, and dissolved gasses and the like, throughout the cell culture media and around the growing cell(s). This is in opposition to well inserts, such as transwell type inserts, which aim to partition a portion of the well to allow the use of more than one cell culture medium, or to provide a directional exposure to a cell culture medium.

[0021 ] Some embodiments of the cell culture insert of the present invention achieve immediate fluid communication between the upper surface and lower surface of the cell growth platform by including a cell growth platform which is fluid permeable. Multiple means are envisaged for providing a fluid permeable cell growth platform(s). In some embodiments, the at least one cell growth platform includes a series of apertures. In some embodiments, the at least one cell growth platform includes, or is provided by, a framework of bars. These bars can be arranged in parallel or can be crossed. [0022] In addition to being fluid permeable, it may be advantageous for the at least one cell growth platform to allow transmission of light. This is particularly advantageous when transmitted-light microscopy is used. Therefore, in some embodiments, the at least one cell growth platform permits transmission of light. This can be achieved by the same means used for fluid permeability as discussed above or may be achieved by alternative means such as using a light transparent material to form the cell growth platform.

[0023] In some embodiments, to allow the cell(s) provided on the at least one cell growth platform to be enveloped by cell culture medium, the at least one cell growth platform includes a series of projections which define the lower surface and wherein fluid can flow between the series of projections and above the defined lower surface. This allows fluid to flow above the cell, or cell cluster, positioned on the lower surface of the growth platform. These projections may comprise a framework of bars that project to define the lower surface.

[0024] The at least one cell culture platform is retained in position by way of at least one well insert. In some embodiments, the at least one well insert is provided by an elongate arm, or multiple elongate arms.

[0025] In some embodiments, the at least one well insert is provided by a sidewall which attaches to at least a portion of the periphery of the at least one cell growth platform. The sidewall may be an inverted frustum sidewall with the at least one cell growth platform positioned at the narrow end of the sidewall. Or the sidewall may be a cylindrical sidewall with the at least one cell growth platform located at one end of the cylinder. In embodiments including a sidewall, it may be advantageous if the end opposite the at least one cell growth platform is open. This can permit the addition of fluid to the upper surface of the at least one cell growth platform, which (in embodiments where the upper and lower surfaces of the at least one cell growth platform are in immediate fluid communication) will flow into the well in which the insert is positioned.

[0026] In some embodiments, where the well insert includes a sidewall, the sidewall includes an aperture which extends upward from the at least one cell growth platform, wherein the aperture permits fluid to flow between the upper and lower surface of the cell growth platform. This allows for immediate fluid communication between the upper and lower surface of the cell growth platform, and may permit access of fluid handling devices to aspirate fluid from a well.

[0027] The present invention further provides a cell culture system including a cell culture insert as described above and a multi-well plate. The system may also include a cover configured to fit over the cell culture insert. In some additional embodiments, the cover is configured to fit over the cell culture insert and the multi-well plate.

[0028] As discussed above, one advantage of the present invention may be the enablement of high-throughput visualisation from below the cell culture plate. Therefore, in some embodiments, each well of the multi-well plate has a light transmissible bottom, preferably an optically transparent bottom.

[0029] To minimise refraction of light when the cell, or cell cluster, is viewed through the bottom of the well, some embodiments of the cell culture system include a multiwell plate wherein the bottom of at least one well of the multi-well plate has a flat portion, or preferably the entire bottom of at least one well of the multi-well plate, is flat.

[0030] The present invention further provides a method of culturing at least one cell, the method including the steps of: embedding the at least one cell in a solid, or semisolid, cell supporting scaffold; providing a cell culture insert including, at least one well insert adapted for insertion into a well of a multi-well plate, and at least one cell growth platform attached to the at least one well insert; applying the at least one cell embedded in the cell supporting scaffold to the lower surface of the at least one cell growth platform; and positioning the at least one well insert into a well of a multi-well plate such that the defined lower surface of the at least one cell growth platform is positioned at a predetermined distance from the bottom of the well. The method may further include the step of providing cell culture media to a well of the multi-well plate.

[0031] Preferably, the cell culture insert used in the method is the cell culture insert described herein.

[0032] The method of the present invention can be used for the culture of any suitable cell(s) such as a cell cluster. However, in some preferred embodiments, the method is used for the culture of a spheroid, a three-dimensional cell aggregate, an embryoid body or an organoid. In a most preferred embodiment, the method is used for the culture of an organoid.

[0033] Organoids can be derived from a range of cell sources and may form a range of model organ systems. Accordingly, in some embodiments of the present invention the organoid is a brain organoid or a stem-cell derived organoid.

[0034] Solid and semi-solid cell supporting scaffolds which can be used for cell culture are known in the art. The viscosity of solid and semi-solid scaffolds acts to localise and orient the cell, or cell cluster, in culture. Preferred forms of the solid and semi-sold cell supporting scaffolds include secreted extracellular matrices, natural extracellular matrices (which may be secreted) and/or synthetic extracellular matrices.

[0035] As discussed above, the cell insert of the present invention may be useful in simplifying visualisation of cell(s), or cell clusters, grown in culture. Therefore, the present invention provides a method of visualisation of at least one cell, the method including performing the method of cell culture of the invention described herein, wherein at least one well of the multi-well plate has a light transmissible bottom and visualising the at least one cell through the bottom of a well in the multi-well plate. Preferably, the light transmissible bottom of the multi-well plate is a light transparent bottom.

[0036] The method of visualisation can be and suitable visualisation technique, or may use any suitable equipment, but is preferably microscopy. In some embodiments of the method of visualisation, the microscopy includes vertical-light microscopy, transmitted-light microscopy and fluorescent microscopy.

Brief Description of Drawings

[0037] Embodiments of the invention will now be described with reference to the accompanying drawings. It is to be understood that the embodiments are given by way of illustration only and the invention is not limited to or by these illustrations.

[0038] Figure 1 illustrates a first embodiment of the cell culture insert of the present invention (cell culture insert 1). [0039] Figure 2 illustrates a second embodiment of the cell culture insert of the present invention (cell culture insert 2).

[0040] Figure 3 illustrates a third embodiment of the cell culture insert of the present invention (cell culture insert 3).

[0041] Figure 4 illustrates a fourth embodiment of the cell culture insert of the present invention (cell culture insert 4).

[0042] Figures 5A to D illustrate close-ups of four exemplified embodiments of the well insert and cell growth platform of the present invention (5A=cell culture insert 1 , 5B=cell culture insert 2, 5C=cell culture insert 3 and 5D=cell culture insert 4).

[0043] Figures 6A to D illustrates the bottom view of the well inserts and growth platforms of four exemplified embodiments of the present invention (6A=cell culture insert 1 , 6B=cell culture insert 2, 6C=cell culture insert 3 and 6D=cell culture insert 4).

[0044] Figure 7 illustrates a cell culture system having a multi-well plate, a cell culture insert and a lid.

[0045] Figure 8 illustrates an exemplified timeline used for growing brain organoids using the exemplified culture inserts of the present invention.

[0046] Figure 9 illustrates immunostaining of brain organoids grown without tissue culture inserts (control) or using one of the four exemplified cell culture inserts (inserts 1 to 4). PAX6: a marker for forebrain and radial glial cell (VZ/SVZ), TLIJ1 (TLIBB3): marker for neurons (CP), TBR1 & TBR2: markers for intermediate progenitors (IZ), MAP2: marker for neurons dendrites (CP); SOX2: marker for radial glial cells (VZ). DAPI: nuclear dye. VZ: ventricular zone, SVZ: subventricular zone, IZ: intermediate zone, CP: cortical plate.

[0047] Figure 10 illustrates live imaging of brain organoids grown on different cell culture inserts over time (days 11 to 56). Images were taken approximately every 3.5 days using a InCell Analyzer 2200 automated fluorescence microscope equipped with a 2.1x objective. A. Organoid grown using cell culture insert 1 (as illustrated in Figure 1); B. Organoid grown using cell culture insert 2 (as illustrated in Figure 2); C. Organoid grown in using cell culture insert 3 (as illustrated in Figure 3); D. Organoid grown using cell culture insert 4 (as illustrated in Figure 4). E. Organoids grown in the absence of a cell culture inserts.

[0048] Figure 11 Illustrates a comparison of brain organoid growth rates using the exemplified cell culture inserts (inserts 1 to 4) vs no inserts.

[0049] Figure 12 illustrates the results of RNAseq analysis showing the expression of different brain region genes in brain organoids derived from H9 (lines 1 ,3, 4, 5, 6) and H9 GFP (lines 8-11) cells grown without a cell culture insert (no insert) or with one of the four exemplified cell culture inserts (inserts 1 to 4). For comparison RNAseq analysis of undifferentiated 2D stem cell cultures of H9 (lines 2) and H9 GFP (Line 7) hESC were also compared. Expression levels (TMM Iog2 normalized counts) are pseudocolored from each gene in each condition from -2.5 to 2.5 as represented in the colour bar.

Detailed Description

[0050] For a further understanding of the aspects and advantages of the present invention, reference should be made to the following detailed description and examples, taken in conjunction with the accompanying figures which illustrate certain embodiments of the present invention.

[0051 ] Tissues develop in vivo in complex three-dimensional matrices which results in nutrient, oxygen and energy gradients as well as cell-to-cell interactions and cell-to- matrix interactions. Traditional two-dimensional cell culture fails to replicate such an environment. Therefore, typically, such two-dimensional techniques are unsuitable for studying the complexities of organ development and the effects of interventions on an organ. To address these limitations a variety of organotypic cell culture systems have been developed in the art that better mimic physiological microenvironments. These cell culture systems are useful in a range of environments and industries, such as the pharmaceutical industry, and permit accurate and meaningful observation of cell differentiation and tissue development.

[0052] Typical three-dimensional organotypic cell culture platforms include hanging drop cell culture (see for example US 2014/0179561 A1 and Eder T and Eder I. 2017, Methods Mol Biol. 1612:167-175), culture of cells on non-adherent surfaces, spinner flask and rotary cell culture system (see for example Lancaster M and Knodlich J. 2014, Nat Protocols. 9(10):2329-2340 and Tang Y. et al. 2017, Sci Reports. 7: art192). Other more simplistic organotypic cell culture systems merely require providing cells in suspension in an appropriate cell media and allowing for cell aggregation.

[0053] These traditional three-dimensional culture systems are typically not high- throughput, and most of these “bulk” cell culture systems will not allow the tracking of individual cell clusters, let alone individual structures or cells within a given cell cluster.

[0054] To try and address some of these problems, various microfluidic (organoids on a chip) devices have also been developed to increase spheroid formation efficiency (for example see Fan Y. et al. 2016, Sci Reports. 6:25062 and Karzbrun E et al. 2018, Nat Physics. 14:515-522). Many of these systems, however, still suffer from problems when culturing cells long term, and are not compatible with many desired interventions and treatments. In addition, many of these microfluidic devices are not compatible with various existing high-throughput screening techniques, such as microscopy, and thus are not suitable for rapidly assessing the effects of interventions and treatments on cell clusters in culture.

[0055] As set out above, the present invention is predicated, in part, on the development of an in vitro cell culture platform which allows for high-throughput processing and screening of cells in culture. Specifically, the methods and culture systems of the present invention allow organotypic culture of cells, and cell clusters, which permits the formation of complex biological tissues in a manner that replicates, in part, the normal physiology and function of the tissue. This includes cell differentiation and aggregation into three-dimensional structures which physiologically resemble native organs.

[0056] As used herein, the term “organotypic cell culture” and “three-dimensional cell culture” refers to cell culture techniques, systems and platform that allows for the development of three-dimensional cell clusters or cell aggregates within a cell culture system. These clusters or aggregates may consist of homogenous cells but will more likely consist of heterogenous cells. In some forms the cell aggregates form differentiated cells which separate and self-organise to form complex structures which mimic miniature organs. [0057] Organotypic cell cultures allow for the assessment of interactions between different cell types, as well as observing the growth of complex biological tissues, in a way that replicates part of their normal physiology and function.

[0058] Importantly, the cell culture inserts, cell culture systems and methods of the present invention allows for organotypic cell culture in manner that is suitable for high- throughput screening. Use of the present invention results in the restriction of the location and orientation of a cell cluster within a cell culture system. Consequently, this permits more rapid visualisation of the cell cluster as well as allowing the observation of specific cells or features within the cell cluster for prolonged periods of time (e.g. weeks, months). Maintaining the position of cells, cell clusters or cell aggregates with respect to the plate permits high-resolution imaging for cell tracking, and allows for relocation of the cell culture between an incubator and a visualisation stage without the relative position of the cell cluster changing within the cell culture system.

[0059] In one aspect, the invention provides a method of culturing a cell, or a cell cluster, the method including the steps of: embedding at least one cell in a solid, or semi-solid, cell supporting scaffold; providing a cell culture insert including at least one well insert adapted for insertion into a well of a multi-well plate and the cell growth platform defining a lower surface and an upper surface, the at least one cell growth platform attached to the at least one well insert; applying the at least one cell embedded in the cell supporting scaffold to the lower surface of the at least one cell-growth platform; positioning the cell culture insert into a well of a multi-well plate such that the defined lower surface of the cell growth platform is positioned at a predetermined distance from the bottom of the well; and optionally providing cell culture media to a well of the multi-well plate. In particular, the method of the invention relates to three- dimensional or organotypic cell culture.

[0060] The method of the present invention permits growth of at least one cell, or a cell cluster, at a predetermined distance from the bottom of a well in a multi-well plate, without being in contact with the bottom of the well. This is facilitated by embedding the at least one cell, or a cell cluster, in a cell-supporting scaffold which attaches the at least one cell to the lower surface of the cell growth platform and maintains the location and orientation of the cell, or cell cluster, over time. [0061] Locating the cell, or cell cluster, on the lower surface of the cell growth platform, at a predetermined distance from the bottom of a well, allows for rapid localisation of the cell or cell cluster during visualisation and avoids the need for prescan or automated localization functions.

[0062] In some embodiments, the predetermined distance of the lower surface of the cell growth platform from the bottom of a well is from 0.5mm up to 4mm, or up to 3.5mm, or up to 3mm. In some preferred embodiment the predetermined distance of the lower surface from the bottom of a well is from 1mm to 2.5 mm, most preferably around 2.5mm. Ultimately, the predetermined distance from the bottom of the well will be determined by the user and will be influenced be a range of technical parameters such as: the type of cell, or cell cluster, being cultured, the intended period of cell culture and therefore the terminal age and size of the cell or cell cluster, the imaging system being used (e.g. wide-field fluorescence, confocal and multiphoton microscopy) and the required objective working distance of lens(es) used for imaging.

[0063] In some embodiments, the at least one cell may multiply to form a cell cluster, or a cell cluster may be formed and embedded into the cell supporting scaffold before being applied to the lower surface of the cell supporting platform. In such embodiments, the cell supporting scaffold orients the cell cluster in a manner that allows for continued visualisation of a specific cell or features within the cluster. This may be particularly advantageous when monitoring the development of a complex three- dimensional cell cluster over multiple time points (e.g. seconds through to months) and may allow for studies of cell migration and differentiation using techniques such as timelapse or video microscopy.

[0064] The cell supporting scaffold (for embedding one or more cells) also allows cells to aggregate to form, or to develop into, a cell cluster as the cells adopt, or differentiate to, their native in vivo function. A “cell cluster” as used herein, encompasses any plurality of cells, whether aggregated or dispersed. Examples of cell clusters can be found in Kenny P et al., 2007, Mol. Oncol., 1 (1): 84-96. In some embodiments, the cluster of cells may be in the form of a spheroid, a compact aggregate, a loose aggregate, or as a suspension of cells in the cell supporting matrix. Other forms of cell clusters are contemplated. In some embodiments, the cells cluster is in the form of a tissue-like structure such as an “organoid”. In some embodiments, the embedded at least one cell is a single cell such as a pluripotent stem cell, an oocyte, a differentiated cell or a cancer cell.

[0065] Adherent cells have an inherent tendency to aggregate to form compact spheres termed “spheroids”. Spheroids are typically rounded, with smooth peripheries and mimic various aspects of solid tissues and are equipped with inherent gradients for diffusion of gases (such as oxygen) and nutrients as well as the removal of metabolic waste. Spheroids typically form from aggregates of differentiated cells such as ex vivo tumour cells and provide a model to study the development of those cells into three- dimensional structures as well as studying their interaction with specific matrices and other cell types, such as non-cancerous cells and vascular cells. Spheroids can be generated from a wide range of cell types and common examples include embryoid bodies, mammospheres, tumour spheroids, hepatospheres and neurospheres. Accordingly, in some embodiments the cell cluster is a spheroid. In some embodiments, the cell cluster is a three-dimensional cell aggregate. In some embodiments, the cell cluster is an embryoid body.

[0066] Organoids are distinguishable from spheroids as they are typically grown from non-terminally differentiated multipotent, totipotent or pluripotent cells including stem cells, embryonic stem cells and induced stem cells. During organoid development, cells differentiated and self-assemble to form complex microenvironments which mimic tissues and organs in vivo. Consequently, organoid cell cultures can provide opportunities for studying the development of complex tissues as well as the physical environment that drive the development and differentiation of cells. As such, they provide ideal candidates for more accurately modelling how organs and tissues will respond to interventions such as a drug treatment.

[0067] Organotypic cell culture for studying a wide range of tissues and organs are reviewed in Shamir ER and Ewald AJ. 2014, Nat Rev Mol Cell Biol. 15; 647-664. Furthermore, methods for preparing and utilising three-dimensional cell culture systems are provided in Soker S and Skardal A (eds) 2018, Tumor Organoids, Humana Press, Cham; Kasper C, Charwat C, Lavrentieva A (eds) 2018, Cell Culture Technology, Springer, Cham; and Haycokc J 2014, 3D Cell Culture, Humana Press. [0068] Organotypic cell culture can be used to develop organs and tissues including (but not limited to): mammary glands, salivary glands, kidneys, lungs, the small intestine, the colon, the liver, the stomach, the pancreas, the oesophagus, the skin, the prostate, the optic cup, the brain and blood vessels. Cellular sources for use in organotypic cell culture include (but are not limited to): cell lines, primary cells, whole organs, tissue organoids, embryonic whole organs, embryonic stem cells, induced pluripotent cells, neoplastic cells and organ slices. Accordingly, in some embodiments, the method of the present invention includes culturing a cell cluster which includes cells derived from mammary glands, salivary glands, kidneys, lungs, the small intestine, the colon, the liver, the stomach, the pancreas, the oesophagus, the skin, the prostate, the optic cup, the brain or blood vessels. In some embodiments, the tissue is diseased tissue or cells, particularly neoplastic or pre-neoplastic tissues or cells.

Cell supporting Scaffolds

[0069] The three-dimensional growth in organotypic cell cultures are facilitated by use of a cell supporting scaffold, which is utilised in the cell culture methods disclosed herein. As its name suggests, the cell supporting scaffold is a medium which supports the maintenance, viability, growth, and replication of a cell in the culture platform. In this regard, the cell supporting scaffold is typically a three-dimensional scaffold which mimics the extracellular matrix (ECM) component of cellular environments found in vivo.

[0070] The ECM performs several critical functions. It provides a complex, nanoscale architecture of structural proteins such as collagen, laminin, and fibronectin to create the mechanical properties inherent in the cellular microenvironment. Cells sense these mechanics through their cell surface integrins and bind to specific adhesion motifs present on the ECM proteins. Cell adhesion in a three-dimensional system leads to and influences a series of subsequent cellular responses that are more physiologically relevant compared to cells grown on 2D surfaces. Furthermore, the ECM is vital for sequestering soluble biomolecules and growth factors and releasing signalling molecules with spatial-temporal control to guide processes such as cell migration, matrix degradation and deposition. Remodelling of the ECM is imperative for achieving tissue homeostasis and is particularly pronounced during development and diseases. Therefore, the cell supporting scaffold must exhibit the mechanical and biochemical properties of in vivo ECM, not only at the initial stage of cell seeding, but also, in a dynamic and tuneable manner as the cells grow and develop.

[0071] The three-dimensional nature of the cell supporting scaffold allows flow of critical elements such as nutrients, gases, salts, growth factors and other micro- and macro-molecules to the cells as well as permitting cell migration therethrough. Suitable cell supporting scaffolds for use in the methods of the invention, or for use with the cell culture inserts and cell culture systems disclosed herein, are known in the art, for example see Dhaliwal A 2012, Mater Methods, 2:162 (D0l//dx.doi.org/10.13070/mm.en.2.162), Panwar A et al. 2016, Molecules, 21 : 685; and Aljohani W et al., 2018, Int J Biol Macro., 107(Pt A): 261-275.

[0072] In some embodiments, the cell supporting scaffold is a solid cell supporting scaffold. In some embodiments, the cell supporting scaffold is a semi-solid cell supporting scaffold. In some embodiments, the cell supporting scaffold is a secreted extracellular matrix. In some embodiments, the cell supporting scaffold is natural, such as a natural extracellular matrix. In some embodiments, the cell supporting scaffold is synthetic, such as a synthetic extracellular matrix. Suitable matrices for forming the cell supporting scaffold are disclosed in the art including Lee J. et al. 2008, Tissue Eng Part B Rev. Mar;14(1):61-86; and Edmondson R etal. 2014, Assay Drug Dev Technol. 2014 May 1 ; 12(4): 207-218

[0073] In some embodiments, the cell supporting scaffold is a gel which may contain a gel or gel-like material. In some embodiments, the gel is a hydrogel. Hydrogels are comprised of networks of cross-linked polymer chains or complex protein molecules of natural or synthetic origin. Due to their significant water content, hydrogels possess biophysical characteristics very similar to natural tissue, and serve as highly effective matrices for three-dimensional cell culture. Hydrogels can be used as standalone three-dimensional matrices or combined with other technologies, such as: bioinks (which can also be used as the hydrogel), solid scaffolds, permeable supports, cellular microarrays, and microfluidics devices. The morphology, growth and functionality of cells within a hydrogel matrix depends on the presentation of biophysical and biochemical cues, as well as physical properties such as permeability and matrix stiffness. [0074] In some embodiments, the cell support scaffold may be a natural or a synthetic scaffold. In some embodiments, the cell support scaffold is a thermosensitive scaffold hydrogel, a photosensitive scaffold, an ionic polymerisation scaffold, an irreversible gelling scaffold, an enzymatic, covalent, or noncovalent polymerisable scaffold, or a cross-linked scaffold.

[0075] Naturally derived scaffold for cell culture are typically formed of proteins and/or ECM components, including scaffolds from Corning Inc., Sigma Aldrich, Trevigen, Inc., NovaMatrix and Xylyx Bio. These scaffolds include components such as gelatin, collagen, laminin, fibrin (the combination of fibrinogen and thrombin - thrombin is used to rapidly polymerize fibrinogen to form fibrin - see Duong H et al., 2009, Tissue Engineering Part A, 15(7): 1865-1876), hyaluronic acid, chitosan, basement membrane extract (Cultrex®), alginate or silk. Derived from natural sources, these gels are inherently biocompatible and bioactive. They also promote many cellular functions due to the presence of various endogenous factors, which can be advantageous for supporting viability, proliferation, function, and development of many cell types.

[0076] A common natural cell supporting scaffold is Matrigel. Matrigel is an ECM- based natural hydrogel that has been used extensively for three-dimensional cell culture in vitro and in vivo. This reconstituted basement membrane is extracted from Engelbreth-Holm-Swarm (EHS) mouse tumors and contains common ECM molecules found in basement membrane (i.e. , laminin, collagen IV, heparin sulfate proteoglycan, and nidogen/entactin). The ECM components of Matrigel scaffold activate various signalling pathways in cancer cells that control angiogenesis, cancer cell motility, and drug sensitivity. Accordingly, in some embodiments, the cell supporting scaffold is Matrigel.

[0077] Collagen Type I is a common ECM molecule found in stromal compartments and bone. It can be isolated from various biological sources including bovine skin, rat tail tendon, and human placenta. Collagen I can also be electrospun into membranes and can support three-dimensional cell growth and differentiation.

[0078] Other naturally derived scaffolds encompassed by the present invention include cell-derived ECM scaffold (e.g. TissueSpec®) and de-cellularised matrix derived from tissue. These scaffolds are isolated from specific tissues or organs which preserve the biomolecules from the original tissues (see Pati F et al., 2014, Nature Comm., 5: 3935; and Choudhury D et al., 2018, Trends Biotechnol., 36(8): 787-805).

[0079] Other natural three-dimensional scaffolds that may be used for forming the cell supporting scaffold include matrices developed in organisms that are animal-free or derived from recombinant nucleic acid technology. Hyaluronic acid (hyaluronan or HA) is one such biologically derived matrix (Gurski LA etal., 2009, Biomaterials, 30(30): 6076-6085). Most commercial grade HA is of bacterial origin and characterized by high purity and homogeneous quality.

[0080] Gelatin methacryloyl (GelMA) hydrogels may also be used and comprise gelatin modified by replacing amino groups to methacryloyl groups to become photo cross-linkable. GelMA has excellent thermostability and its solid-liquid transition is influenced by temperature (Sun M et al., 2018, Polymers, 10(11): 1290). The photocrosslinked form is crosslinked by adding initiator under UV light.

[0081] As indicated above, the cell support scaffold may be synthetic. Synthetic scaffolds may be chosen when naturally-derived biological matrices are unsuitable. Synthetic scaffolds are comprised of purely non-natural molecules (see DeVolder R and Kong HJ, 2012, Wiley Interdiscip. Rev. Syst. Biol. Med., 4(4): 351-365) such as poly(ethylene glycol) (PEG) (Sawhney AS et al., 1993, Macromolecules, 26(4): 581- 587), poly(vinyl alcohol)(Martens P and Anseth KS, 2000, Polymer, 41 (21): 7715- 7722), poly(2-hydroxy ethyl methacrylate) (Chirila TV et al., 1993, Biomaterials, 14(1): 26-38), Polyglycolic acid (PGA) (Xu H et al. 2010. Int J Med Robot., 6:66-72), poly(methyl methacrylate) (PMMA), Polylactide-co-glycolide (PLGA) Zhu X et al. 2008, Biotechnol Bioeng. 100:998-1009) and Pluronic F127 thermosensitive synthetic hydrogel. They are biologically inert but provide structural support for various cell types.

[0082] PEG scaffolds have been shown to maintain the viability of encapsulated cells while allowing for ECM deposition as the scaffold degrades, thereby demonstrating that synthetic gels can function as an appropriate culture platform in the absence of integrin-binding ligands. Such inert gels are highly reproducible, allow for facile tuning of the mechanical properties, and are simple to process and manufacture.

[0083] It is also possible to modify inert synthetic cell support scaffolds with appropriate biological components. Examples of synthetic hydrogels that are tunable include Pura Matrix™ (Corning), VitroGel®, VitroGel® 3D, VitroGel® 3D-RGD, and VitroGel® 3D-RGD-PLUS (TheWell Bioscience, North Brunswick, New Jersey, USA), TrueGels (Sigma-Aldrich) and peptide hydrogel.

Cell Culture Media

[0084] In addition to the cell culture scaffold, which supports the three-dimensional growth of the cell clusters, three-dimensional cell culture typically requires a cell culture medium. Cell culture medium is typically a liquid designed to support the growth of cells, and generally includes an energy source and compounds for regulating the cell cycle. A typical culture medium will include a complement of amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones, and attachment factors. In addition to nutrients, the medium also helps maintain pH and osmolality. Cell culture media can be specifically designed to drive differentiation and development of specific cell types and will be tailored to the indented use.

[0085] The methods of the present invention include submerging, or otherwise exposing, the at least one cell embedded in the cell supporting scaffold in an appropriate cell medium, or media. The cell culture medium may be any medium which is capable of maintaining the viability and/or growth of a cell or is capable of transporting or delivering required components to the cell supporting scaffold without affecting the cells viability. Exemplary media include those available from commercial sources such as ThermoFisher Scientific (Gibco Cell Culture Media), HyClone™ Cell Culture Media, and Sigma Aldrich (including DMEM - Dulbecco's Modified Eagle Medium, DMEM/F12, Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12, Ham's F-10 Nutrient Mixture, Ham's F-12 Nutrient Mixture, Media 199, MEM, Minimum Essential Media RPMI Medium 1640, Advanced Media, Opti-MEM I Reduced Serum Media IMDM, Iscove's Modified Dulbecco's Medium, Gibco Cell Culture Bags, FluoroBrite DMEM Media). Suitable cell culture media is also available from StemCell Technologies (including mTeSR™ Plus, mTeSR™, mTeSR™1 , MethoCult™ H4034 Optimum, BrainPhys™ Neuronal Medium, IntestiCult™ Organoid Growth Medium (Mouse), AggreWell™ EB Formation Medium, Agar Leukocyte Conditioned Medium), LONZA media (including OGMTM, Osteoblast Growth Medium SingleQuotsTM Supplements and Growth Factors, EGMTM -2 MV, Microvascular Endothelial Cell Growth Medium-2 BulletKitTM, EGMTM Endothelial Cell Growth Medium BulletKitTM, EGMTM-MV Microvascular Endothelial Cell Growth Medium BulletKitTM, MEGMTM Mammary Epithelial Cell Growth Medium BulletKitTM, MBMTM-4 Melanocyte Growth Basal Medium-4, LGM-3TM Lymphocyte Growth Medium-3, FGMTM Fibroblast Growth Medium BulletKitTM, FGMTM-2 Fibroblast Growth Medium-2 BulletKitTM, PGM-2TM Preadipocyte Growth Medium-2 BulletKitTM, KGMTM Gold Keratinocyte Growth Medium BulletKitTM, tEGMTM Retinal Pigment Epithelial Cell Growth Medium BulletKitTM, B-ALITM Bronchial Air-Liquid Interface Medium BulletKitTM, SAGMTM Small Airway Epithelial Cell Growth Medium BulletKitTM, KGMTM-CD Keratinocyte Growth Medium BulletKitTM- Chemically Defined, SCGMTM Stromal Cell Growth Medium BulletKitTM, KGMTM-2 Keratinocyte Growth Medium-2 BulletKitTM, Calcium Free, AGMTM, Astrocyte Growth Medium BulletKitTM, MsGMTM Mesangial Cell Growth Medium BulletKitTM, ABMTM Astrocyte Basal Medium, SkGMTM-2 Skeletal Muscle Cell Growth Medium-2 BulletKitTM, BEGMTM Bronchial Epithelial Cell Growth Medium BulletKitTM, PrEGMTM Prostate Epithelial Cell Growth Medium BulletKitTM, SmGMTM- 2 Smooth Muscle Cell Growth Medium -2 BulletKitTM etc) and PromoCell (including Endothelial Cell Growth Medium, Fibroblast Growth Medium, Adipocyte Nutrition Medium, Airway Epithelial Cell Growth Medium, Chondrocyte Growth Medium, Keratinocyte Growth Medium, Mammary Epithelial Cell Growth Medium, Melanocyte Growth Medium, Osteoblast Growth Medium, Pericyte Growth Medium, Skeletal Muscle Cell Growth Medium, Smooth Muscle Cell Growth Medium, Small Airway Epithelial Cell Growth Medium).

[0086] Appropriate cell culture media, and methods of customising cell culture media are known in the art. For example, see Arora M, 2013, Mater Methods, 3:175.

Visualisation of Cell Cultures.

[0087] An advantage of the methods of organotypic cell culture disclosed herein are that they allow for high-throughput screening of cells or cell clusters. One important aspect of screening is visualisation of the cells or cell clusters. Therefore, the present invention provides a method of visualisation of a cell cluster, the method including the steps of: embedding at least one cell in a solid, or semi-solid, cell supporting scaffold; providing a cell culture insert including; at least one well insert adapted for insertion into a well of a multi-well plate; and at least one cell growth platform defining a lower surface and an upper surface, the at least one cell growth platform attached to the at least one well insert; applying the at least one cell embedded in the cell supporting scaffold to the lower surface of the at least one cell growth platform; positioning the cell culture insert into a well of a multi-well plate such that the defined lower surface of the at least one cell growth platform is positioned at a predetermined distance from the bottom of the well; and optionally providing cell culture media to a well of the multi-well plate, wherein at least one well of the multi-well plate has a light transmissible bottom, and visualising the cell, or cell cluster, through the bottom of a well in the multi-well plate. In some embodiments, the light-transmissible bottom is a light transparent bottom.

[0088] Suitable materials are known in the art for forming, or providing, light- transmissible and light-transparent biocompatible cell culture plates. In this context, biocompatible should be considered as compatible with cell survival and cell growth.

[0089] In some embodiments, particularly when light-transmission microscopy is utilised, it is advantageous for the cell growth platform to permit the transfer of light through the cell growth platform. Therefore, in some embodiments, as will be further discussed below, the cell growth platform is light-translucent or light-transparent.

[0090] Methods are known in the art for visualisation of cells growing in cell culture. In some embodiments of the method of visualisation, the visualisation is vertical-light microscopy or transmitted-light microscopy. In some embodiments, the visualisation is fluorescent microscopy. Specific microscopy techniques for visualising cell culture, including three-dimensional cell culture, include (but are not limited to): bright-field microscopy, phase-contrast microscopy, two-photon and multi-photon excitation microscopy, confocal microscopy, fluorescence microscopy, fluorescence recovery after photobleaching (FRAP), Forster Resonance Energy Transfer (FRET), fluorescence lifetime imaging microscopy (FLIM), total internal reflection fluorescence (TIRF), super-resolution microscopy, differential interference-contrast microscopy (DIG), video-enhanced DIG microscopy.

Cell culture inserts

[0091] As defined above, the present invention provides a cell culture insert, for use with a multi-well plate, the cell culture insert including: at least one well insert adapted for insertion into a well of a multi-well plate; and at least one cell growth platform defining a lower surface, the at least one cell growth platform supported by the at least one well insert such that the lower surface of the at least one cell growth platform, when positioned within a well of a multi-well plate, is positioned at a predetermined distance from the bottom of a well.

[0092] Embodiments, of a cell culture insert in accordance with the present invention are illustrated in an in use position in Figures 1 to 4 and are described further below.

[0093] A reference to an “in use” orientation as used herein refers to the orientation of the cell culture insert 1 during culturing. During preparation of the cell culture, particularly during application of cells to the growth platform, the cell culture insert 1 may be inverted so that the cell growth platform is facing upward. This allows the cell supporting scaffold to set, solidify or polymerise prior to cell culture.

Cell culture insert embodiment 1

[0094] Figure 1 illustrates a first embodiment of the cell culture insert 1 of the present invention (in an in use orientation) for use with a 24-well cell culture plate. The cell culture insert 1 includes 24 frustum shaped well inserts 2 (see further Figure 5) which terminate at the lower end with a horizontal cell growth platform 3. The cell growth platform 3 is provided by a solid circular surface providing a planar lower surface (in use).

[0095] The well insert 2 is provided by a continuous side wall which surrounds the circumference of the cell growth platform 3 and extends vertically to attach to a planar support 4. The support 4 is configured to abut and be supported by the upper surface of a multi-well plate (see Figure 7). Consequently, the cell growth platform 3 is maintained at a predetermined distance from the bottom of a well of a cell culture plate.

[0096] The planar support 4 includes two apertures 5 on the lateral sides of the well insert 2. As such, the well insert 2 comprises a traversely-elongate frustum which traverses a well of a 24-well cell culture plate. The apertures 5 are configured to permit fluid to be added to a well and/or removed from a well when the insert 1 is positioned in a multi-well plate. This allows a user to change, or modify, cell culture medium during incubation without having to remove the cell culture insert 1. This is particularly advantageous when high-throughput fluid handling is used.

Cell culture insert embodiment 2

[0097] Figure 2 illustrates a second embodiment of the cell culture insert 1 of the present invention for use with a 24-well cell culture plate. The embodiment of Figure 2 is similar to embodiment 1 illustrated in Figure 1 , with the exception of the cell growth platform 6.

[0098] The cell growth platform 6 of the second embodiment includes a series of downward-projecting parallel bars 7 which form a framework and define the lower surface of the cell growth platform 6. The parallel bars 7 (see Figures 5B and 6B) are interspaced with recessed portions 8 which allow fluid flow. Consequently, when a cell, or cell cluster is positioned (within a cell support scaffold) on the lower surface of the cell growth platform 6 fluid can flow between the series of projecting bars 7 and above the defined lower surface thereby allowing fluid to envelop the cell supporting scaffold and the cultured cells. As such, the cell growth platform 6 of embodiment 2 is configured to allow fluid communication around a cell positioned on the lower surface of the cell platform.

Cell culture insert embodiment 3

[0099] Figure 3 illustrates a third embodiment of the cell culture insert 1 of the present invention for use with a 24-well cell culture plate. The cell culture insert 1 includes 24 well inserts. Each well insert is provided by two elongate arms 9, which downwardly converge toward the supported horizontal cell-growth platform 10 (see Figure 5C). While the embodiment of Figure 3 comprises two elongate arms, which are converging, the well insert may be provided by a single elongate arm, or more than two elongate arms.

[0100] The cell growth platform 10 of embodiment 3 is further illustrated in Figure 6C. As can be seen, the cell growth platform 10 is provided by a series of parallel bars 11 , and has a lower surface and an upper surface (not shown in Figure 6C). The bars 11 are interspaced by slots 12 and an annular opening making the cell growth platform 10 fluid permeable which allows immediate fluid communication between the lower surface and the upper surface of the cell growth platform 10.

[0101] As used herein the term “immediate fluid communication” means that fluid can freely flow between the upper and lower platform. When used in cell culture, with the cell culture insert 1 inserted into a well of a cell culture plate, culture medium added to the well is free to immediately flow between the lower surface and the upper surface of the cell growth platform 10. Inserts, such as transwell type inserts are made to limit the flow of fluid from the bottom surface of the microporous membrane to the upper surface, thereby allowing different mediums to be placed in the lower/outer well provided by the culture plate and in the internal well provided by the positioned transwell insert. Further, transwell type cell culture systems include a microporous membrane which can allow selective diffusion of molecules (such as hormones, growth factors and drugs), cell-to-cell interaction across the membrane, angiogenesis across the membrane, or migration and invation of specific cell types across the membrane, while still maintaining separation of some cells or media between the internal well and lower/outer well. These conditions are not required by the present invention.

[0102] While the embodiment of Figure 3 utilises a framework of parallel bars and slots, alternative means are envisaged for providing a fluid permeable cell growth platform. These include, but are not limited to, a cell growth platform that includes a series of apertures. For example, a planar cell growth platform such as that illustrated in Figure 1 may include a series of aperture which will permit fluid and light to traverse the cell growth platform. Alternatively, the framework of bars may be crossed bars (e.g. a mesh).

[0103] In addition to allowing immediate fluid communication, the slots 12 provided between the parallel bars 11 allow for light to traverse the cell growth platform 10. This is particularly advantageous when using light-transmission microscopy where the cell(s) is/are illuminated from the top and the optics are positioned at the bottom.

[0104] The cell growth platform 10 also includes a raised peripheral annular rim 15 which provides a well, which is inverted when in use. This helps locate a cell supporting scaffold, and its corresponding cell(s), on the cell growth platform 10. In the exemplified embodiment of Figures 3, 5C and 6C the peripheral annular rim 15 is about 1 mm thick and acts as a barrier to assist in localising the position of the cell, cell cluster or cell aggregate.

[0105] The two elongate arms 9 of the well insert, provide a large top aperture 13, and two lateral apertures 14 that extend upward from the cell growth platform. These apertures 14 permit fluid to be added to a well and/or removed from a well when the cell culture insert 1 is in use with a multi-well plate, thereby allowing a user to change, or modify, cell culture medium during incubation. Further, the lateral apertures 14 permit fluid to flow between the lower surface of the cell growth platform and the upper surface thereby enhancing the immediate nature of the fluid communication.

Cell culture insert embodiment 4

[0106] Figure 4 illustrates a fourth embodiment of the cell culture insert 1 of the present invention for use with a 24-well cell culture plate. The cell culture insert 1 includes 24 well inserts 16 provided by a cylindrical side wall that extends from a support 4 downward to the cell growth platform 17. The cylinder has an open top end (not shown) and terminates at the bottom end with a cell growth platform 17 which includes a framework of parallel bars 11 interspaced by slots 12 (see Figures 5D and 6D). As described above for the cell culture insert of embodiment 3, the slots allow for immediate fluid communication between the lower surface and the upper surface of the cell growth platform 17, allowing medium to envelope an embedded cell, or cell cluster during culturing.

[0107] Further, the cell culture platform includes a raised peripheral annular rim 15 (also see Figures 5D and 6D) which provides a well, which is inverted when in use. This helps locate a cell supporting scaffold, and its corresponding cell(s), on the cell growth platform 17.

[0108] The open top end of the cylinder, in combination with the slots 12 allow light to traverse the cell growth platform in the manner described above for the cell culture insert of embodiment 3.

[0109] A planar support 4 is provided that includes two semi-circular apertures 5 per support which are spaced apart by a rectangular portion which attaches the well inserts 18 to the support. [0110] The cell culture inserts illustrated in Figures 1 to 4 are configured for use with 24-well cell culture plates. However, it is to be understood that the inserts can be readily adapted to be used with plates comprising fewer or more wells. In some embodiments, the cell culture insert includes a single well insert and corresponding cell growth platform. In some embodiments, the cell culture insert includes multiple well inserts and corresponding cell growth platforms. In some embodiments, the cell culture insert includes two well inserts and corresponding cell growth platforms. In some embodiments, the cell culture insert includes four well inserts and corresponding cell growth platforms. In some embodiments, the cell culture insert includes eight well inserts and corresponding cell growth platforms. In some embodiments, the cell culture insert includes 12 well inserts and corresponding cell growth platforms. In some embodiments, the cell culture insert includes 48 well inserts and corresponding cell growth platforms. In some embodiments, the cell culture insert includes 54 well inserts and corresponding cell growth platforms. In some embodiments, the cell culture insert includes 96 well inserts and corresponding cell growth platforms.

[0111] In the illustrated embodiments of the cell culture insert (Figures 1 to 6) the cell growth platform is suspended at a distance of 19.5mm from the planar support 4 to provide a predetermined distance of 2.5mm from the bottom of the well of a 22mm deep multi-well plate. This distance allows work with organoids throughout their entire development and imaging using wide-field fluorescence, confocal and multiphoton microscopy.

[0112] Moreover, the diameter of the growth platform in the exemplified embodiments, at its lowest point, is 3 mm. While other diameters are envisaged (or widths when the cell growth platform is not circular) a diameter of 3mm allows the cell(s), or cell cluster, to maintain their position during growth while permitting the removal and replacement of cell culture medium by way of liquid handling equipment. Further, a diameter of about 3mm allows for the inserts to be suitable for use in standard 24-well and 48-well cell culture plates while still permitting fluid handling.

[0113] The cell culture insert of the present invention can also be used in combination with a multi-well plate to provide a cell culture system. Such a system is illustrated in Figure 7 as a longitudinal cross-section. The system of Figure 7 illustrates the cell culture insert of embodiment 1 in cooperation with a 24 well plate 19. As can be seen, the well inserts 2 extend downwardly into the wells 20 of the multi-well plate 19. The support 4 abuts the upper surface of the multi-well plate 19, to maintain a predetermined distance between the bottom of the wells 20 and the cell growth platforms 3. The system further includes a cover 21 configured to fit over the cell culture insert and over the multi-well plate 19 thus helping reduce potential contamination of the cell culture.

[0114] The culture insert and system, and its various structural parts such as the multi-well plate, may be manufactured using any suitable method known in the art. These include standard moulding techniques (e.g. injection moulding or precision glass moulding), three-dimensional printing, stereolithography, selective laser sintering, fused deposition modelling, lithography or combinations thereof. The technique chosen will be determined by a range of factors including the material being used, the size and scale of the system and cost considerations.

[0115] A variety of materials suitable for forming the cell culture inserts and systems disclosed herein are known in the art and include any suitably ridged material such as plastics, metals, silica oxides, silica glasses and quartz. Suitable plastic include (but are not limited to) acrylonitrile butadiene styrene (ABS), polydimethylsiloxane (PDMS), polyimide, polybutylene, polypropylene, polycarbonate, poly(methyl methacrylate) (PMMA), cyclic olefin copolymer, polyethylene, polyethylene terephthalate, polyurethane, polycaproleacton, polyactic acid, polyglycolic acid, or poly(lactic-co- glycolic acid), Acrylic, OrdylSY330, polyaryletherketone, (PEKK), fluorinated ethylene propylene (FEP), fluoropolymers PTFE (polytetrafluoroethylene) and PFA (perfluoroalkoxy polymer resin), Flexdym™ and polystyrene such as tissue culture polystyrene (TOPS). For example, see Lerman MJ et al. 2018 Tissue Eng Part B Rev:, 24(5): 359-372, Ryan JA, 2008 BioFiles, 3.8, 21 ; van Midwoud PM et al. 2012 Anal. Chem. 2012 May 1 ;84(9):3938-44; and Halldorsson S et al., 2015 Biosens. Bioelectron., 63: 218-231 , which provide disclosures of relevant materials and their sources. The material from which the cell culture inserts and the cell culture system (including the multi-well plates) are made from is not limited so long as the material is biocompatible or suitable for purpose. This can be assessed by a person skilled in the art in view of their knowledge. [0116] As the cell culture insert and system of the present invention are required to be sterile, the materials from which they are formed need to be suitable for sterilization. In some embodiments, plates are sterilized prior to use or packaging. Sterilization is performed using any method suitable for the material of the plate and the intended use. These include (but are not limited to) heat and/or high pressure, chemicals (e.g. ethanol, ethylene oxide), irradiation (such as gamma irradiation) or combinations thereof.

[0117] The cell culture insert, the multi-well plate and lid of the cell culture system, may be made of the same or different material. Further, some components of the insert, such as the growth platform, may be made of different material, or material having different properties, to other components. For example, the cell growth platform may comprise a visually transparent or translucent material while the well insert may be visually opaque. Additionally, the bottom of the wells of the cell culture plate of the cell culture system may be visually transparent or comprised of a specific optical material such as silica, while the side walls of the wells, or the body of the multi-well plate, may be formed of opaque material or lower cost material.

[0118] In preferred embodiments, the bottom of the well(s) of the plate, for use in the cell culture system, are substantially visually transparent (transparent and colourless, transparent coloured, or translucent).

[0119] Some aspects of the invention utilize commercially available multi-well plate. Multi-well plates are commercially available from manufacturers or distributers such as Corning, Millipore, Becton-Dickinson, Eppendorf, CellVis and PerkinElmer amongst others.

[0120] As discussed, an advantage of the present invention is that the method, cell culture insert and system of the present invention are useful for high-throughput screening (HTS). As such, it is advantageous for the cell culture inserts, and systems, to be compatible with microscopy, analytical, and/or automated systems that are used in drug discovery and relevant fields of chemistry and biology. The present invention may allow researchers to perform larger number of tests in a day compared to traditional techniques. Mainstream HTS instruments are designed to perform operations or tasks, such as liquid handling, imaging, microscopy, or optical detection, on samples contained on a multi-well plate that complies with ANSI/SBS standards. In some embodiments, the system of the invention complies with standards, for example ANSI/SBS standards, therefore allowing the device to be used with HTS instruments which means the generation and assessment of organotypic cell cultures can be scaled up. The apertures provided in the supports, and/or between the well inserts, allow liquid to be easily transferred in and out of the wells for maintenance and treatment of the embedded cell(s) or cell cluster(s).

[0121] Organotypic cell culture can be used for a range of research, clinical and screening applications which include (but are not limited to):

• Predictive assays for screening therapeutic candidates such as potential cancer drugs. The inability to screen potential drugs and drug combination in individual patients limits the development of drug in physiological systems relevant to humans, or specifically to individual conditions. Organotypic cell culture methods and systems, such as those disclosed herein, when used with high-throughput screening allow for the rapid assessment of a range of potential therapeutics using physiologically relevant tissues such as ex vivo slice cultures from primary tumours or ex vivo primary tumour cells. These can be studied in combination with other supporting tissues such as vascular tissues. In this regard, the one or more therapeutic agents may be selected from the group consisting of a drug, a small molecule, a nucleic acid, an oligonucleotide, an oligopeptide, a polypeptide, a protein, an enzyme, a polysaccharide, a glycoprotein, a hormone, a receptor, a ligand for a receptor, a co-factor, an antisense oligonucleotide, a ribozyme, a small interfering RNA, a microRNA, a short hairpin RNA, a lipid, an aptamer, a virus, and nanoparticle and an antibody or an antigen binding part thereof. Particularly envisaged predictive assays include screening of drug candidates for treatment of cancer

• Prognostic assays for measuring functionally based outputs including morphological criteria (e.g. cell migration and invasion), molecular criteria (e.g. gene and protein expression);

• Preclinical therapeutic testing to provide proof-of-concept testing of potential of compounds to regulate and manage disease outcomes; • Genome studies using genetic tools such a zinc-finger nucleases (ZFN)Transcription activator-like effector nucleases (TALEN) or Cluster of Regularly- Interspaced Short Palindromic Regions (CRISPR) to study genetic influences of tissue and organ development and cell differentiation as well assessing potential genetic therapies;

• Developmental analysis using pluripotent or multipotent cells such as embryonic stem cells, induced pluripotent cells, embryos, fused embryos or blastocysts. By incubating these cells in organotypic cell culture systems the development and differentiation of tissues can be studied in real time;

• Rational therapeutic design to assess the interaction of therapeutic candidates and the functionality of therapeutics within a three-dimensional multicellular tissue or organ, thereby allowing for rational development of drug regimens;

[0122] In view of the above, in some embodiments of the present invention the cell culture inserts, systems and the methods disclosed herein may be used for drug discovery and toxicity testing; culture of embryos, tissue slices, small organisms, and worms (e.g., c. elegans); and bacteria culture (minimal contact with surface prevents biofilm formation and increases gas exchange, which is important for many types of bacteria).

Examples

Materials and Methods for Preparing and Culturing Brain Organoids using cell culture inserts

[0123] Brain organoid development using the cell culture insert described in embodiments 1 , 2, 3 and 4 was compared to organoid development in the absence of cell inserts.

[0124] Brain organoids were prepared in accordance with the protocol set out in Lancaster and Knoblich (Nature Protocols. 2014 Oct; 9(10):2329-40), with minor modifications as set forth below, in accordance with the timeline illustrated in Figure 8.

[0125] T-25 flasks were prepared by coating low growth factor Matrigel (Corning - catalogue number 356230) using a solution of Matrigel at a concentration of 10 pl Matrigel in 3 ml of DMEM/F12 culture media (Invitrogen - catalogue number 11330- 032). The Matrigel containing flasks were then incubated at 37°C for 1 hour before seeding. Matrigel solution was then removed from the T-25 flask by washing with 2ml of Dulbecco’s PBS (DPBS), before 4 ml of pre-warmed mTESRI media (Stem Cell Technologies - catalogue number 05850) containing 8 pl of a 5 mg in 2.96 ml of water dilution of RHO/ROCK pathway (ROCK) inhibitor Y27632 (Stemcell Technologies - catalogue 72304).

[0126] The human embryonic stem cell (hESC) line H9, or H9 cells expressing green fluorescent protein (H9GFP), were partially thawed in a water bath before being transferred to 4 ml mTESRI media in a 15 ml tube. The hESCs were pelleted by centrifugation at 200 x g for 5 minutes before the supernatant was removed. The hESCs were resuspended in 2 ml of mTESRI medium, then transferred into the pre-prepared T-25 flasks and distributed evenly by gentle shaking. The plated cells were maintained by daily replacement of 4 ml to 5 ml of mTESRI media.

[0127] When the hESCs prepared as per the protocol above were ready for splitting (approximately 80% confluent) they were either cultured to form embryoid bodies, split (passaged) or frozen.

[0128] To split cells, once cells were 80% confluent (typically 5 to 7 days after seeding) the cells were split to new prepared T-25 flasks (prepared as above). Briefly, hESCs were washed twice with 1 ml to 2 ml of EDTA or DPBS at 37°C. One ml of EDTA working solution (0.5 mM; calcium and magnesium free in DPBS) was then added to the T-25 flasks before incubation at 37°C for 4 minutes. The EDTA was removed and 2 x 3 ml of mTESRI medium (Stem Cell Technologies - catalogue number 05850) was added to form a cell suspension. One ml of the 6 ml cell suspension was transferred to a prepared T-25 flask containing 3 ml of mTESR medium before the flasks were returned to incubate at 37°C. Cells were used fresh to form embryoid bodies, or frozen for later use.

Making Embryoid Bodies

[0129] Eighty percent confluent hESCs prepared as set out above were washed once with 2 ml PBS before 1 ml of EDTA (0.5 mM in calcium and magnesium free DPBS) was added. This was followed by incubation at 37°C for 4 min. Following aspiration of the EDTA solution, 1500 l of Accutase (Sigma catalogue number 6964) was added to the cells in the T-25 flask and incubated on ice for 3 minutes. One thousand, seven hundred pl of mTESR media was added to the flask to form a single cell suspension before 3 ml of the cell suspension was transferred to a 15 ml conical tube and a cell count was performed on the remaining 200 pl to calculate the number of cells in the 3 ml cell suspension.

[0130] Cells were pelleted by spinning the conical tube at 600 x g for 4 minutes before the supernatant was aspirated. Cells were resuspended to a concentration of 1.280⁵ cells/ml in filtered low bFGF hES medium (40 ml DMEM/F12, 10 ml KOSR, 1.5 ml stem cell quality FBS, 0.5 ml GlutaMAX, 0.5 ml MEM-NEAA and 100 ul of 50mM 2- ME). Four ng/ml bFGF (1 :2500) and 100 pM ROCK inhibitor (1 :100 dilution of the 5 mg in 2.96 ml stock solution) were added and 150 pl of the cell suspension was deposited in each well of a low adhesion 96 well Il-bottom multi-well cell culture plate. Cells were maintained by changing the Low bFGF hES medium every 3 days (without bFGF and ROCK inhibitor) until embryoid bodies were formed.

Making Primitive Neuroepithelia

[0131] When embryoid bodies reached a diameter of 500 to 600 pm in thickness and begun to brighten and show smooth edges (approximately 5 to 6 days after addition of cells to wells), the embryoid bodies were transferred to 400 pl ml filtered (0.22 pm) neural induction medium (100 ml DMEM/F12, 1 ml N2 supplement, 1 ml GlutaMAX supplement, 1 ml MEM-NEAA and 100 pl Heparin solution) in low-cell binding 24-well plates. Embryoid bodies were maintained by changing the neural indication medium every second day, until visible neuroepithelia appeared (approximately 4 to 5 days). Embryoid bodies were observed to ensure that they had smooth edges and brightening of their periphery, indicating viable embryoid bodies

Example 1 - Making Cerebral Tissue Brain Organoids Using the Organoid Cell Culture System

[0132] Cell culture inserts of embodiments 1 , 2, 3 and 4 were prepared by sterilising the insert in 80% v/v ethanol, before drying in an aseptic biosafety cabinet. The insert was then exposed to UV light for 30 minutes. [0133] Once primitive neuroepithelia were formed (as set out above), they were embedded in 200 pl of Regular Matrigel (Corning - catalogue number 354234) and positioned on the upward facing surface of an inverted cell culture insert (lower surface in use) using a modified p1000 pipette tip. An inverted multi-well plate was then placed over the insert to protect the Matrigel embedded neuroepithelia and the Matrigel was left to polymerise for 20 minutes at 37°C.

[0134] A 24 well culture plate was prepared with 400 pl of neural induction medium. Once the Matrigel polymerised the insert was returned to the in use orientation and the embedded neuroepithelia were positioned in an individual well of a multi-well plate and incubated at 37°C.

[0135] Two days later the cell medium was aspirated and replace with 400 pl of Improved Differentiation Medium-A (125 ml DMEM/F12, 125 ml Neurobasal, 1.25 ml N2 supplement, 5 ml B27- vitamin A supplement, 62.5 pl insulin, 250 pl of 50 mM 2-ME solution, 2.5 ml GlutaMAX supplement, 1.25 ml MEM-NEAA and 2.5 ml penicillinstreptomycin) containing 3 pM CHIR99021 (Sigma-Aldrich catalogue number SML1046-5MG), diluted in DSMO.

[0136] Three days after the addition of the Improved Differentiation Medium-A + CHIR9902, the medium was replaced with Improved Differentiation Medium-A (without CHIR9902) and incubated for a further 2 days before the developing organoids were feed with new medium and the cell culture plates were transferred to an orbital shaker. Two days subsequently, the organoids were fed further with Improved Differentiation Medium+A (125 ml DMEM/F12, 125 ml Neurobasal, 1.25 ml N2 supplement, 5 ml B27- vitamin A supplement, 62.5 pl insulin, 250 pl of 50 mM 2-ME solution, 2.5 ml GlutaMAX supplement, 1.25 ml MEM-NEAA and 2.5 ml Penicillin Streptomycin) supplemented with 1 mg of NaHCOs. Cells were fed with Improved Differentiation Medium+A supplemented with 1 mg of NaHCOs every 3 to 4 days up until day 40 (22 days on the orbital shaker), when the medium was changed over to Improved Differentiation Medium + 1 :50 regular Matrigel + 1.5 mg/ml NaHCOs. Organoid culture continued until analysis was performed.

Example 2 - Making Control Cerebral Tissue Brain Organoids. [0137] To compare brain organoids grown using the cell culture system of the present invention to those grown in the absence of the cell culture inserts, brain organoids were grown using the protocol set out above, with the following exception. Once primitive neuroepithelia formed, they were embedded in Regular Matrigel and left to polymerise for 20 minutes at 37 °C. The embedded cells were then placed in 400pl of neural induction medium in a well of 24-well cell culture plate which had flat and clear bottoms, and incubated at 37°C. The embedded cells were then processed as above until analysis was performed.

Example 3 - Immunofluorescent Analysis of Brain Organoids

[0138] Organoids prepared using the cell culture insert as described above, were fixed in 4% paraformaldehyde for 30 minutes at room temperature and then washed with PBS before being dehydrated with 30% sucrose for 16 hours at 4°C.

[0139] Fixed organoids were embedded in a solution of 10% sucrose and 7.5% gelatine and frozen in -50°C isopentane. Frozen embedded organoids were serially sectioned at a thickness of 20 pm in a cryostat and mounted on slides. Slides were stored at -80°C until use.

[0140] Mounted organoid sections were washed with PBS and then incubated in 0.2% permeabilisation solution at room temperature for 30 minutes. After being washed in PBS three times, the sections were incubated in 5% Bovine Serum Albumin (BSA) blocking solution at room temperature for 1 hour. Each section was then incubated at 4°C for 16 hours with 100 pl of diluted primary antibody prepared in 5% BSA blocking solution to the concentration set forth in Table A.

Table A - Primary Antibody Concentration

[0141] Sections were then washed with PBS before being incubated with an appropriate fluorescent-conjugated secondary antibody, prepared in 1% BSA solution, for 1 hour at room temperature.

[0142] Prior to imaging, stained sections were cured with Prolong™ Diamond Antifade Mountant (ThermoFisher Scientific - catalogue number P3665) at room temperature for 1 hour. Imaging was performed on a Zeiss LSM700 confocal microscope.

[0143] Figure 9 illustrates immunofluorescent staining against different cortical markers of brain organoids grown on the cell culture insert of embodiments 1 , 2, 3 and 4 as well as those grown in the absence of a cell culture insert (control). Specific markers illustrated include: PA12 - a marker for forebrain and radial glial cells in the ventricular zone (VZ) and subventricular zone (SVZ); TLIJ1 (TLIBB3) - a marker for neurons found in the cortical plate (CP); TBR1 & TBR2 - markers for intermediate progenitor cells found in the intermediate zone (IZ); MAP2 - a marker for neuron dendrites found in the CP; SO9 - a marker for radial glial cells found in the VZ; and DAPI - a nuclear dye.

[0144] As can be seen in Figure 9, there were no notable differences in the cellular organization and differentiation of organoids grown on the cell culture inserts compared to those grown in the absence of inserts. Further, there were no distinguishable difference in the formation of different brain cortical structures (ventricular zone, subventricular zone, intermediate zone and cortical plate). Consequently, it can be concluded that use of the cell culture insert described herein do not adversely alter the development and differentiation of organoids such as brain organoids.

Example 4 - Live Organoid Imaging

[0145] Brain organoids were prepared from the H9 GFP stem cell line using the protocol described above on cell culture inserts 1 , 2, 3 and 4. A control group of organoids were grown in an identical manner except for the absence of a cell culture insert. [0146] An InCell Analyzer 2200 (GE Healthcare Life Sciences) equipped with GFP fluorescence filter cubes and Differential Interference Contrast (DIC) was used to image brain organoids. Images were collected using a 2.1 x objective lens every 3.5 days between day 11 and day 39 with a further image collected on day 56 using 0.1 second exposure time in the GFP channel and 0.1 second exposure time in the DIC channel.

[0147] Images from the entire plate were analysed using an Imaged macroinstruction plugin which imported all the images of the plate into a stack, before GFP fluorescence was used for segmentation of the organoids. Green fluorescent protein mean fluorescence and organoid size was measured from the segments across each well at the various time points. Data generated by the Imaged plugin were then exported and analysed using Microsoft Excel and Graphpad Prism.

[0148] Figure 10 shows representative images of brain organoids grown on the various cell culture inserts (cell inserts of embodiments 1 , 2, 3 or 4) or in the absence of a cell culture insert. Figure 11 shows a graphical representation of the growth rate of brain organoids grown on different cell culture inserts or in the absence of a cell culture insert. All data points represent mean ± standard error for at least 3 organoids per insert at each given time point.

[0149] As can be seen in Figure 10, and as quantified in Figure 11, the rate of growth of the brain organoids in the cell culture system using cell culture inserts 1 , 2, 3 or 4 (Figures 10A, B, C and D, respectively) is comparable to the rate of growth of brain organoids grown in the absence of a cell culture insert (Figure 10E). Importantly, however, the orientation of the brain organoids grown on the cell culture inserts remained stable as can be seen by the consistent location of the notable features indicated by the arrow in Figures 10A, B and C. By comparison, the conformation of the brain organoids grown in the absence of a cell culture insert (Figure 10A) was different for each time point.

[0150] By providing the organoids in a consistent orientation, interventions performed on the organoids can be more accurately analysed and assessed because any data generated will not be influenced by changes in organoid orientation. Further, the development of particular features, or differentiation of movement of specific cells within the organoid can be tracked over time as the position of the organoid is maintained.

[0151] Additionally, the consistent positioning of the organoid relative to the bottom of the well (i.e. in the z axis), and from the sides of the well (i.e. in the x and y axes), makes locating the organoid for image capturing considerably easier. This enables preprogrammed automated image capturing without pre-scan or without automated localization functions as the location of each organoid within the cell culture system is known. It can also allow for continued time-lapse imaging as the location of the organoid is fixed.

Example 5 - mRNA Profile of Brain Organoids

[0152] To analyses the ability of the cell culture inserts of the present invention to develop functional brain organoids, the gene expression of specific brain markers was analysed in brain organoids grown using the technique described above using cell culture inserts 1 , 2, 3 and 4. These brain organoids were compared to organoids grown on no insert and undifferentiated H9 and H9GFP cells grown in the absence of a cell supporting scaffold (i.e. grown in 2D).

[0153] Once the brain organoids were developed, total RNA was isolated using TRIzol reagent (Invitrogen - catalogue number 15596026) in accordance with the manufacturer’s instructions. Extracted RNA was then purified with Monarch® RNA Cleanup Kit (New England Biolab - catalogue number T2040S) and analysed for quality in the Agilent 2100 Bioanalyzer and confirmed to have an RNA Integrity Number greater than 8. The quantity of RNA extracted was calculated in a Qubit 4 Fluorometer (Invitrogen).

[0154] RNA-seq was used to profile the transcriptome of brain organoids grown on cell culture inserts 1 , 2, 3 or 4, in the absence of a cell culture insert or in the absence of both Matigel and a cell culture insert (2D growth). To perform RNA-seq analysis, 300 ng of RNA was used to generate RNA-seq transcriptome libraries using KAPA standard RNAseq HyperPrep kit (Kapa Biosystems) and the quality of the library was validated with an Aglent 2100 Bioanalyser. RNA-seq libraries from organoids derived from H9 and H9GFP cell lines grown using the different tissue culture inserts were multiplexed and sequenced on two separate runs using the Illumina NextSeq 500 platform and the stranded single end protocol with a read length of 75 base pairs. Sequenced reads derived from both runs were merged for each sample before further processing. Raw data, averaging 70.5 million reads per sample were analysed and quality-checked using the FastQC program (http://www.bioinformatics.babraham.ac.uk/projects /fastqc). The STAR spliced alignment algorithm (version 2.5.3a with default parameters) was used to map the sequence reads against the human reference genome (hg19) (see Dobin, A. et al., Bioinformatics. 2013; 29:15-21), returning an average unique alignment rate of 90%. The gene counts were TMM normalised using R (version 3.2.3) and edgeR³ (version 3.3). Matlab was used to analyse alignment and check quality of sequence data.

[0155] Figure 12 shows the expression of different brain region maker genes in brain organoids derived from H9 (columns 1 , 3, 4, 5, 6) and H9GFP (columns 8 to 11) cells grown in the absence of a cell culture insert (“no insert”) or on one of cell culture inserts 1 , 2, 3 or 4 (columns 3, 4, 5, 6, 8, 9, 10 and 11). For comparison, RNA-seq analysis was performed on undifferentiated 2D cell cultures of H9 (column 2) and H9GFP (column 7) stem cells. Expression levels (TMM Iog2 normalized counts) are coloured from each gene in each treatment from -2.5 to 2.5 as represented in the colour bar.

[0156] Figure 12 illustrates that the gene expression profile of marker genes for different brain regions were similar in the brain organoids grown using cell culture inserts 1 , 2, 3 or 4 when compared to brain organoids grown in the absence of an insert.

[0157] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0158] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises”, is not intended to exclude other additives, components, integers or steps. [0159] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.

[0160] While the present invention has been described with reference to specific, and in some instances preferred, embodiments it is to be understood that the invention is not limited to the disclosed examples. The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combined or claimed with one or more other compatible feature(s) of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.

[0161] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.

[0162] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

[0163] Also, it is to be noted that, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context already dictates otherwise.

Claims (47)

The claims defining the invention are as follows:

1. A cell culture insert, for use with a multi-well plate, the insert including: at least one well insert adapted for insertion into a well of a multi-well plate; and at least one cell growth platform defining a lower surface, the cell growth platform supported by the at least one well insert such that the lower surface of the cell growth platform, when positioned within a well of a multi-well plate, is positioned at a predetermined distance from the bottom of a well.

2. The cell culture insert of claim 1 , wherein the predetermined distance from the bottom of the well is from 0.5mm to 4mm, or is from 0.5mm to 3.5mm, or is from 0.5mm to 3mm, or is from 1 mm to 2.5 mm, or is about 1 ,5mm, about 2mm or about 2.5mm.

3. The cell culture insert of claim 1 or claim 2, wherein the at least one cell growth platform has a width or diameter of from 2mm to 4mm, or from 2.5mm to 3.5mm or about 3mm.

4. The cell culture insert of any one of claims 1 to 3, wherein the lower surface of the at least one cell growth platform is configured to allow fluid communication around a cell positioned on the lower surface of the cell platform.

5. The cell culture insert of any one of claims 1 to 4, wherein the at least one cell growth platform includes an upper surface and a lower surface, the upper and lower surfaces in immediate fluid communication when the cell culture insert is in use with a well of a multi-well plate.

6. The cell culture insert of any one of claims 1 to 5, wherein the cell culture insert includes a support for abutting and being supported by a multi-well plate.

7. The cell culture insert of claim 6, wherein the at least one well insert is attached to the support.

8. The cell culture insert of claim 7, wherein the support is attached to multiple well inserts, each well insert having a corresponding cell growth platform.

9. The cell culture insert of claim 8, wherein the support includes 12, 24, 48 or 54 well inserts.

10. The cell culture insert of any one of claims 6 to 9, wherein the support abuts and is supported by an upper surface of a multi-well plate.

11. The cell culture insert of any one of claims 6 to 10, wherein the support, the at least one well insert and the at least one growth platform are configured to provide an aperture to permit fluid to be added to a well and/or removed from a well when the insert is positioned in a multi-well plate.

12. The cell culture insert of any one of claims 1 to 11 , wherein the at least one cell growth platform includes a series of projections which define the lower surface and wherein fluid can flow between the series of projections and above the defined lower surface.

13. The cell culture insert of any one of claims 1 to 12, wherein the at least one cell growth platform permits transmission of light.

14. The cell culture insert of any one of claims 1 to 13, wherein the at least one cell growth platform is fluid permeable.

15. The cell culture insert of any one of claims 1 to 14, wherein the at least one cell growth platform includes a series of apertures.

16. The cell culture insert of any one of claims 1 to 15, wherein the at least one cell growth platform includes, or is provided by, a framework of bars.

17. The cell culture insert of claim 16, wherein the bars are arranged in parallel or are crossed.

18. The cell culture insert of claim 16 or claim 17, wherein the framework of bars project to define the lower surface.

19. The cell culture insert of any one of claims 1 to 18, wherein the cell growth platform is horizontal in use.

20. The cell culture insert of any one of claims 1 to 19, wherein the at least one well insert is provided by an elongate arm.

21 . The cell culture insert of any one of claims 1 to 19, wherein the at least one well insert includes multiple elongate arms.

22. The cell culture insert of any one of claims 1 to 19, wherein the at least one well insert is provided by a sidewall which attaches to at least a portion of the periphery of the at least one cell growth platform.

23. The cell culture insert of any one of claims 1 to 19, wherein the at least one well insert is provided by an inverted frustum sidewall with the at least one cell growth platform positioned at the narrow end of the sidewall.

24. The cell culture insert of any one of claims 1 to 19, wherein the at least one well insert is provided by a cylindrical sidewall and the at least one growth platform is located at one end of the cylinder.

25. The cell culture insert of claim 23 or claim 24, wherein the end opposite the at least one cell growth platform is open.

26. The cell culture insert of any one of claims 22 to 25, wherein the sidewall includes an aperture which extends upward from the at least one cell growth platform, wherein the aperture permits fluid to flow between an upper surface and the lower surface of the at least one cell growth platform.

27. The cell culture insert of any one of claims 1 to 26, wherein the at least one cell growth platform includes a peripheral annular rim which, in use, extends downward from the lower surface.

28. A cell culture system including a cell culture insert according to any one of claims 1 to 27; and a multi-well plate.

29. The cell culture system of claim 28, further including a cover configured to fit over the cell culture insert.

30. The cell culture system of claim 29, wherein the cover is configured to fit over the cell culture insert and the multi-well plate.

31 . The cell culture system of any one of claims 28 to 30, wherein each well of the multi-well plate has a light transmissible bottom.

32. The cell culture system of claim 31 , wherein the light transmissible bottom is optically transparent.

33. The cell culture system of any one of claims 28 to 32, wherein a bottom of at least one well of the multi-well plate has a flat portion.

34. The cell culture system of any one of claims 28 to 33, wherein a bottom of at least one well of the multi-well plate is flat.

35. A method of culturing at least one cell, the method including the steps of: embedding at least one cell in a solid, or semi-solid, cell supporting scaffold; providing a cell culture insert including; at least one well insert adapted for insertion into a well of a multi-well plate; and at least one cell growth platform defining a lower surface and an upper surface, the at least one cell growth platform attached to the at least one well insert; applying the at least one cell embedded in the cell supporting scaffold to the lower surface of the at least one cell growth platform; and positioning the at least one well insert into a well of a multi-well plate such that the defined lower surface of the at least one cell growth platform is positioned at a predetermined distance from the bottom of the well.

36. The method of claim 35, further comprising the step of providing cell culture media to a well of the multi-well plate.

37. The method of claim 35 or claim 36, wherein the cell culture insert is the insert of any one of claims 1 to 27.

38. The method of any one of claims 35 to 37, wherein the at least one cell embedded in the cell supporting scaffold is a cell cluster, a spheroid, a three- dimensional cell aggregate, an embryoid body or an organoid.

39. The method of any one of claims 35 to 38, wherein the at least one cell is an organoid.

40. The method of claim 39, wherein the organoid is a brain organoid or a stemcell derived organoid.

41. The method of any one of claims 35 to 40, wherein the solid, or semi-solid, cell supporting scaffold is a secreted extracellular matrix.

42. The method of any one of claims 35 to 41 , wherein the solid, or semi-solid, cell supporting scaffold is a natural extracellular matrix.

43. The method of any one of claims 35 to 40, wherein the solid, or semi-solid, cell supporting scaffold is a synthetic extracellular matrix.

44. A method of visualisation of at least one cell, the method including performing the method of any one of claims 35 to 43, wherein at least one well of the multi-well plate has a light transmissible bottom and visualising at least one cell through the bottom of a well in the multi-well plate.

45. The method of claim 44, wherein the light transmissible bottom is a light transparent bottom.

46. The method of claim 44 or claim 45, wherein the visualisation is vertical-light microscopy or transmitted-light microscopy.

47. The method of any one of claims 44 to 46, wherein the visualisation is fluorescent microscopy.