Cutting device and method of casting use for microfluidic threedimensional cell culture and for tissue mimicking material

Abstract

The present invention describes a device comprising a mould and a cutter, and methods for making them, where the mould is configured to receive one or more substrates, and the cutter may comprise a fluidic component. The device as disclosed herein has particular application in laboratory research.

Description

[0001] Novel Device

[0002] Field of the invention

[0003] The present invention relates to the fields of biotechnology and fluidic devices. In particular, the invention relates to a novel lab-on-chip system which has particular application in research such as in biological research and high-throughput 2D and 3D cell culture.

[0004] Background of the invention

[0005] An increasing body of evidence shows that traditional in vitro and in vivo animal models poorly correlate with human (patho)physiology. In vitro studies do not capture the complexity and dynamicity of human tissues and organs, while in vivo studies raise ethical concerns and are hampered by interspecies differences. These translational challenges currently hinder clinical translatability of results obtained from these models and limit their scientific applicability and societal impact, emphasizing the need for alternatives. 3D tissue models such as organ-on-chip (OoC) systems are emerging as robust and near-physiological alternatives to these traditional models. Remarkably, as evidenced by the 2022 U.S. Food and Drug Administration (FDA) Modernization Act 2.0, (international regulatory agencies have recently begun to allow for in vitro alternatives for animal testing of new therapies, which opens new avenues for the scientific and clinical impact of these emerging systems. Nevertheless, scientific progress and clinical translation in this area are limited by the lack of universal and user-friendly platforms that can be employed for a variety of on-chip 3D tissue models.

[0006] Current 3D on-chip culture platforms face significant limitations due to their reliance on injectable tissue-mimicking materials (TMMs) such as hydrogel precursor solutions for integration into microchambers (Rafiei N, et al. Design of a versatile microfluidic device for imaging precision-cut-tissue slices. Biofabrication. 2022 Jul 19;14(4); Gumuscu B, et al. Compartmentalized 3D Tissue Culture Arrays under Controlled Microfluidic Delivery. Sci Rep. 2017 Jun 13;7(1 ):3381 ; Yu J, et al. Reconfigurable open microfluidics for studying the spatiotemporal dynamics of paracrine signalling. Nat Biomed Eng. 2019 Oct;3(10):830-841 ; Gaharwar AK, et al. 3D Biomaterial Microarrays for Regenerative Medicine: Current State-of-the-Art, Emerging Directions and Future Trends. Adv Mater. 2016 Jan 27;28(4):771-81). Other methods for incorporating TMMs into microchambers or microchannels without direct injection are low-throughput and involve multistep, one-by-one insertion processes (e.g., via 3D bioprinting) (WO0107891 A2). Each sample must be individually handled and inserted, making the process time-consuming and inefficient, particularly for high-throughput screening applications. Furthermore, many existing OoC systems are not capable of directly incorporating whole tissues into the fluidic devices. The common requirement for injectability prevents the use of tissue samples for ex vivo culture within these platforms. This limitation hinders the ability to create realistic 3D tissue environments that closely mimic in vivo conditions. Finally, the extensive training and expensive equipment required to operate these complicated systems further hamper their widespread adoption, particularly in smaller laboratories. This complexitycreates a barrier for smaller research facilities and limits the accessibility, scalability, and clinical translatability of these technologies.

[0007] There is, therefore, a need fora versatile device capable of a more rapid application in the laboratory which is at the same time, compatible with high-throughput processes.

[0008] Summary of the Invention

[0009] In one aspect, there is provided a mould comprising a substrate platform, wherein the substrate platform is configured to retain one or more substrates, and wherein the mould is configured to receive the cutter as described herein.

[0010] In a further aspect, there is provided a lid configured to be reversibly connected to the mould assembly and engage with the tank as described herein.

[0011] In a further aspect, there is provided a frame-cutter which is configured to cut excess material from an external sample.

[0012] In a further aspect there is provided a method of making a substrate, the method comprising: i. casting a substrate into the mould assembly as described herein; and

[0013] ii. optionally, engaging the lid as described herein with the mould.

[0014] In a further aspect, there is provided a cutter comprising a plurality of cutting walls configured to cut a plurality of compartments into a substrate, wherein the cutter comprises a plurality of vessels, wherein each vessel comprises an inlet and an outlet, wherein the vessels are conduits to one or more of the compartments.

[0015] In a further aspect, there is provided a method of making a microfluidic device, the method comprising:

[0016] i. introducing one or more substrates into the mould assembly as described herein.

[0017] ii. connecting the cutter comprising the fluidic component as described herein to the mould assembly of step i.

[0018] In a further aspect, there is provided a method of screening drugs wherein,

[0019] i. the drugs are introduced to the microfluidic device as disclosed herein; and

[0020] ii. the drug interaction is measures and / or imaged.Description of the invention

[0021] Definitions

[0022] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the method. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0023] In this document and in its claims, the verb "to comprise" and its conjugations are used in its nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0024] As used herein, the term "and / or" indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0025] As used herein, with "At least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ... ,etc.

[0026] As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. The term “about” as used herein refers to any values, including both integers and fractional components that are within a variation of up to ±10% of the value modified by the term “about.”

[0027] As used herein the terms “device,” “system,” “microfluidic chips,” “multi-layer, multi-gel microfluidic device,” “organ-on-a-chip,” or “organ-on-chip” are synonymous and refer to the multi-layer, multigel microfluidic devices described herein that can be used to grow and cultivate cells, thus forming a “organ-on-a-chip” system.

[0028] As used herein, the terms “control,” “reference level,” and “reference” are used interchangeably and referto a predetermined value or range, which is employed as a benchmark against which to assess the measured result.As used herein, “formulation” and “composition” can be used interchangeably and refer to a combination of at least two ingredients.

[0029] As used herein, the terms “sample” or “test sample” refers any sample in which the presence and / or level of a target is to be detected or determined or any sample treated with the compositions as detailed herein.

[0030] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0031] Detailed description of the invention

[0032] The present inventors have developed a versatile fluidic device which allows for facile, single-step high-throughput integration and compartmentalization of substrates, such as non-injectable tissues and / or tissue mimicking materials (TMMs). A device according to the invention additionally provides a means for facile production of multi-layered substrate constructs, such as multilayered tissues, which makes tissue culture and screening easier. In instances when a device of the invention is constructed on a standard-sized microscopy slide, analysis is further facilitated because of compatibility with various types of microscopes, including inverted, upright, and fluorescence microscopes, further reducing the need for specialized training and expensive equipment.

[0033] Mould and Substrate

[0034] In one aspect, there is provided a mould comprising a substrate platform, wherein the substrate platform is configured to retain one or more substrates, and wherein the mould is configured to receive the cutter as described herein. The skilled person would understand that the mould, also referred to as the mould assembly, thus comprises an open-top tank or receptacle, the base of which forms the substrate platform, said tank or receptacle being configured to receive and retain one or more substrates. The substrates may be in injectable or non-injectable form, i.e. liquid or solid state, as discussed below. The mould as described herein may be used for facile construction of a complex 3D environment without custom design of the microfluidic chip. Beneficially, placing tissue slices in the receptacle of the mould allows for a facile, high throughput ex-vivo tissue model. In instances when a mould as described herein is constructed on a standard-sized microscopy slide, analysis is further facilitated because of compatibility with various types of microscopes , further reducing the need for specialized training and expensive equipment. Therefore, in some embodiments, the mould is configured for compatibility with a standard-sized microscopy slide. In some embodiments the mould is configured for compatibility with a microscope slide of 75 by 26 mm. In some embodiments the mould is configured for compatibility with a microscope slide of 75 by 25 mm. In some embodiments the mould is configured for compatibility with a microscope slide of 76 by 26 mm. In a further embodiment, the substrate platform is configured for compatibility with a standard-sized microscopy slide. In some embodiments, the substrate platform is configured for compatibility with a microscope slide of 75 by 26 mm. In some embodiments the substrate platform is configured for compatibility with a microscope slide of 75 by 25 mm. In some embodiments thesubstrate platform is configured for compatibility with a microscope slide of 76 by 26 mm. In some embodiments, the mould is at least 70 to 80mm by 20 to 80mm. In some embodiment, the mould measures 75 by 25, 76 by 26, 75.5 by 25 mm, 75 by 50, 76 by 51 , 76 by 52 mm, 127.8 by 85.5 mm 84 by 54 mm, 15 by 15, 15 by 30, 15 by 45, 15 by 60, 15 by 75, 30 by 30, 30 by 45, 30 by 60, 30 by 75, 45 by 45, 45 by 60, 45 by 75, 60 by 60, 60 by 75, or 75 by 75 mm. In some embodiments, the mould comprises a tank or receptacle measuring 60 by 22, 60 by 21 , 60 by 18, 66 by 21 , 66 by 21 , 50 by 20, 49.5 by 19.5, 48 by 18, 48 by 72, 99.2 by 63.1 , 99 by 63, 96 by 60, 9 by 9, 24 by 24, 24 by 54, 24 by 48, 54 by 54 mm. The adaptability of the invention, including the mould and / or receptacle for use as a microfluidic device will be known to the skilled person, for example through EVS-EN ISO 22916-2022.

[0035] In some embodiments substrates are mounted inside of the mould assembly. In a further embodiment, substrates are mounted on the substrate platform of the mould assembly. In some embodiments substrates are prepared externally and placed into the mould assembly. In some embodiments substrates are presented in liquid state to the mould assembly. In some embodiments substrates are presented in liquid state onto the substrate platform. The substrate platform may be configured for embodiments wherein the substrate is preformed to formed within the mould assembly. For example, to produce tissue construct inside the mould assembly, injectable or non-injectable substrates, such as tissue mimicking materials (TMMs), may be used, optionally containing cells, spheroids, organoids, particles, or other additives. These materials may be cast into the mould and shaped into uniform sheets of controlled thickness, optionally using an interchangeable lid system.

[0036] Therefore, in a further aspect, there is provided a lid configured to be reversibly connected to the mould assembly and engage with the tank as described herein. The lid is configured to sit on the substrate platform and assist in casting a defined substrate layer, such as a level substrate layer. For example, in one method the liquefied substrate is deposited onto the substrate platform and the lid is pressed down such that the lid presses the liquefied material to fill the mould. In some embodiments, the lid comprises an overflow outlet where excess material exits. In some embodiments, the lid comprises a cutting or stamp portion configured to cut or stamp the substrate. These patterns may be the same for each sample microchamber, or they may be different to facilitate high-throughput production of a wide variety of patterned / shaped samples. To achieve multi-layered constructs, sequential casting may be performed, where each layer may contain different cell types or chemical additives as required. Combinations of the above are possible, such as placing a tissue slice in between two layers of biomaterials. Tissue interfaces can also be constructed this way.

[0037] Therefore, it is understood that the substrate platform is configured to receive one or more substrates. In some embodiments, the substrate is homogeneous in composition and / or structure. Such an embodiment provides many uniform compartmentalized samples, or microchambers, forhigh throughput screening when cut by the cutter, as disclosed herein. In some embodiments, the substrate is heterogeneous in composition and / or structure. One may refer to such a structure as a “tissue slice”, and such an embodiment provides a “pixelated” scanning of the response of different areas of the tissue slice such as different layers of tissues and materials, such as skin or the bonecartilage interface. Such examples will be known to the skilled person, such as presented in Altunbek M et al. Design and bioprinting fortissue interfaces. 2023 Biofabrication 15:2; Gurkan U A. et al. Engineering Anisotropic Biomimetic Fibrocartilage Microenvironment by Bioprinting Mesenchymal Stem Cells in Nanoliter Gel Droplets. 2014 Molecular Pharmaceutics 11 ;7; and Lin Z et al Osteochondral Tissue Chip Derived From iPSCs: Modeling OA Pathologies and Testing Drugs. 2019 Front. Bioeng. Biotechnol. 7.

[0038] In some embodiments, the tissue slice comprises patient biopsy samples. Such an embodiment can be seen to have excellent application in personalized medicine, such as for pre-screening medicine. In some embodiments multi-layered TMMs are cast into the mould. Such TMMs may comprise hydrogels, which may have a particular application for in vitro tissue models. For example, TMMs may be cast in the mould to specific thicknesses or placed in the mould as flat sheets. The skilled person would understand the adaptability of the mould and lid to produce specific thickness and a multilayered substrate. Such an approach is compatible with a wide variety of TMMs, not only injectable gels that organ-on-chip systems tend to be limited to. However, the skilled person would readily understand the application of injectable gels to the mould. It can be envisioned that the TMMs may contain cells, cellular constructs such as spheroids or organoids, or any other additives, such as particles, suitable forthe current application. For example, the gels may be cast into multiple layers of defined thickness, and each layer or border between layers may comprise different cell types or additives. It will be understood that combinations of the above are possible. For example, wherein a tissue slice is cased between two layers of TMMs. As would be known to the skilled person, tissue interfaces bridge the gap between two dissimilar tissues, usually with physical and biological properties that separate them from the tissues they connect, as discussed above. Thus, the skilled person would thus understand that different tissue interfaces (e.g. bone-cartilage, bonetendon, bone-ligament) may be constructed this way. In some embodiments, the substrate comprises one or more selected from Bone-Cartilage, Bone-Tendon, Bone-Ligament, Muscle-Tendon, Cartilage-Synovium, Mesenchymal-Epithelial, Epidermis-Dermis-Hypodermis, Cornea-Stroma, Blood-Brain Barrier, Blood-Retinal Barrier, Gut Epithelium-Lamina Propria, Lung Alveolar-Endothelial-epithelial, and Cochlear Hair Cell-Neuron.

[0039] However, by eliminating the requirement for injectability, as is common in the art (see Junaid and Hankemeier. OrganoPlate Micro-fluidic Microvessel Culture and Analysis. 2021 Bio-Protocol 11 :13; Jones et al. 2022 Design of an Integrated Microvascularized Human Skin-on-a-Chip Tissue Equivalent Model. Front. Bioeng. Biotechnol. 10; and Ates et al. 2024. ACS Omega 9:41 , 421 OS-42115), the invention enables the use of a diverse range of substrates, such as TMMs and whole tissue samples, overcoming the constraints of existing systems. A device according to the inventionfurther supports the direct incorporation of tissues or tissue models, facilitating the creation of realistic in vitro or ex vivo 3D tissue environments.

[0040] External tissue constructs such as porous scaffolds or tissue slices, including ex vivo biopsy samples, may be directly placed onto the mould for subsequent processing. In a further aspect, there is provided a frame-cutter which is configured to cut excess material from an external sample. The frame-cutter thus ensures that the sample fits into the mould assembly.

[0041] In some embodiments, the substrate comprises natural and / or synthetic compositions. In some embodiments, the substrate comprises metallic compositions. In some embodiments, the substrate comprises biological materials. In a further embodiment, the substrate comprises cells and / ortissue derived material. In some embodiments, the substrate comprises human or non-human derived material. In some embodiments, the substrate comprises dietary products. In some embodiment the substrate comprises cellular derived material. In a preferred embodiment, the substrate comprises one or more cellular derived materials selected from stem cells, osteoblasts, fibroblasts, chondrocytes, neurons, immune cells, endothelial cells, or any derivative thereof. In some embodiment the substrate comprises biological tissues of human or non-human origin. In a preferred embodiment, the substrate comprises one or more tissue derived materials derived from one or more selected from bone, blood, skin, cartilage, fat, xylem, leaf, or any tissue derivative thereof. In a preferred embodiment, the substrate comprises one or more dietary products selected from cheese, meat, vegetables, fruits, sugar, or any derivative thereof. In some embodiment the substrate comprises metallic materials. In a preferred embodiment, the substrate comprises one or more metallic materials selected silver, gold, sodium, iron, or any derivative thereof. In some embodiment the substrate comprises one or more materials selected from Gelatin, Gelatin Methacryloyl, Pluronic, Agar, Agarose, Alginate, Collagen, Fibrin, Matrigel, Hyaluronic Acid, Chitosan, Dextran, Polyethylene Glycol, Polyvinyl Alcohol, Decellularized Extracellular Matrix, Cellulose, Silk, Polylactic Acid, Polyglycolic Acid, Polycaprolactone, Poly(lactic-co-glycolic acid), Polydimethylsiloxane, Polyurethane, Polyvinyl Alcohol, Polyethylene Glycol, Ceramics such as hydroxyapatite, or any derivative thereof. In some embodiment the substrate comprises one or more materials selected from glasses such as bioactive glass, carbon-based materials such as graphene and carbon nanotubes, or any derivative thereof. In some embodiments, the substrate requires mixing the cells with thermoset hydrogels (e.g., solubilized basement membrane preparation (Matrigel®), gelatin methacrylamide (GelMA), gelatin, agar, agarose, carrageenan, inter alia) or light-curable hydrogels (e.g., 4-arm-PEG acrylate) and introducing the combination to the mould assembly. This delivers the cells to the mould allowing continuous growth. In another embodiment, a first cured substrate, such as a hydrogel, comprises uncured spaces comprising one or more of cylindrical, spherical, cubic, or other shaped voids that can be occupied by a population of cells encapsulated in a second cured substrate, such as a hydrogel. In some embodiments, either of the hydrogels can comprise a light- or thermally cured hydrogel including a 4-arm polyethylene glycol acrylate, a gelatinous protein mixture, gelatin methacrylamide (GelMA), gelatin, fibrin, agarose,agar, chitosan, carrageenan, solubilized basement membrane preparation (Matrigel®), among others. In some embodiments, the first cured hydrogel comprises a 4-arm polyethylene glycol acrylate, gelatin, fibrin, or a combination thereof that are curable using UV / visible light. The skilled person is by no means limited by the substrate options exemplified and disclosed herein.

[0042] In a further aspect there is provided a method of making a substrate, the method comprising: i. casting a substrate into the mould assembly as described herein; and

[0043] ii. optionally, engaging the lid as described herein with the mould.

[0044] In some embodiments, there is provided a method of making a multilayered substrate or tissue slice, the method comprising:

[0045] i. casting a substrate into the mould assembly as described herein; and

[0046] ii. engaging the lid as described herein with the mould;

[0047] Hi. removing the lid;

[0048] iv. casting a further substrate into the mould.

[0049] In some embodiments, there is provided a method of making a 3D culture, the method comprising: i. introducing one or more TMMs into the mould as described herein.

[0050] ii. optionally, when more than one TMMs is used, separating each TMM with the lid as described herein.

[0051] In some embodiments, the method as disclosed herein is repeated at least 1 , 2, 3, 4, 5, 6, 7 or more times.

[0052] In some embodiments, the mould assembly is configured to reversibly attach to a cutter as disclosed herein. In some embodiments, the mould is configured to irreversibly attach to the cutter. In some embodiments, the mould is configured to attach to the cutter with a watertight seal.

[0053] Cuter Implement

[0054] In a further aspect, there is provided a cutter comprising a plurality of cutting walls configured to cut a plurality of compartments into a substrate, wherein the cutter comprises a plurality of vessels, wherein each vessel comprises an inlet and an outlet, wherein the vessels are conduits to one or more of the compartments.

[0055] The term “compartments” as used herein takes its meaning from the art and includes discrete microchambers, as described herein. The compartments may be described to receive samples of the substrate when the cutter engages with the mould assembly.

[0056] The term “conduit” as used herein takes its meaning from the art to be a natural or artificial channel through which something (such as a fluid) is conveyed. A conduit for rainwater is a pipe, tube.Therefore, the vessel as described herein will be understood to be a channel though which a fluid is conveyed.

[0057] The “cutter implement” may also be simply referred to as the “cutter”, the two terms are interchangeable and used throughout. Therefore, in a further aspect, there is provided a cutter comprising cutting walls configured to cut the substrate into a plurality of compartments. In some embodiments, the cutter is configured to reversibly connect to the mould. In some embodiment, the cutter is configured to irreversibly connect to the mould assembly In some embodiments, the cutter is configured to connected to the mould with a water-tight seal.

[0058] The substrate is cut into a plurality of compartments, samples, or microcompartments (e.g., squareshaped compartments) by the cutting implement. In some embodiments, the amount of compartments on one chip depends on the chosen size of the compartments, for example, in some embodiments there 8x24 compartments of 2.0 x 2.0 mm, i.e the equivalent of two 96-well plates in numbers of compartments on the substrate platform. In some embodiments, there are 6x16 square compartments of 3.0x3.0 mm (96 total), 6x8 rectangular compartments of 3x6 mm (48 total), or 16x24 square samples of ~1.5x1.5 mm (384 total).

[0059] Therefore, following engagement with the cutter, in some embodiments, the substrate comprises 96 (8x12) square compartments that are 3x3 mm in size and typically 0.25-1 mm thick. As would be clear to the skilled person, the design is adaptable to accommodate different shapes and numbers of samples if desired. Further miniaturization is possible, mainly limited by the thickness of the cutting walls. For example, a chip that contains 48 rectangular samples of 3x6 mm, 192 (8x24) square samples of 2x2 mm, or 384 (16x24) square samples of ~1.5x1.5 mm. In some embodiments, there is provided the cutting implement as described herein, wherein the cutter is configures to cut at least 48 compartment, at least 96 compartments, at least 384 compartments into the substrate.

[0060] As can be appreciated, by streamlining the on-chip integration and compartmentalization process, a device according to the invention enables high-throughput screening by allowing simultaneous incorporation of multiple compartments, which may be seen as samples, without the need for tedious, one-by-one insertion of substrate into individual compartment or microchambers. A device according to the invention thus not only improves the efficiency and applicability of organ-on-chip (OoC) systems, but also has the capability of increasing accessibility for smaller laboratories, promoting widespread adoption and advancing the field of 3D tissue modelling. In some embodiments, the cutter has an open top. Such an embodiment allows for composition to be delivered to each compartment or microcompartment by any means already known to the skilled person.The cutter may be patterned to cut or stamp specific structures into the substrate to make discrete samples. These patterns may be the same for each compartment, or they may be different, to facilitate high-throughput production of a wide variety of patterned / shaped compartment. To achieve multi-layered constructs, sequential casting is performed, with each layer containing different cell types or chemical additives as required. Combinations of the above are possible, such as placing a tissue slice in between two layers of biomaterials. Tissue interfaces can also be constructed in this way. External tissue construct such as porous scaffolds or tissue slices, including ex vivo biopsy samples, may also be directly introduced into the mould assembly for subsequent processing, or any combination of external tissue and injectable substrate is also within the scope of the invention. As stated above, beneficially, various tissues or TMMs that are cuttable may be incorporated into the chip, in contrast to conventional microfluidic chips which are limited to injectable materials.

[0061] In some embodiments, there is provided the cutting implement as described herein, wherein the cutting walls are configured to partially cut the substrate or wherein the cutting walls are configured to fully cut the substrate. As discussed further below, in some embodiments, the rows of microchambers are isolated from each other. In some embodiments, the rows of microchambers are connected to each other. Such embodiments may be adjusted though the height of the cutting walls. For example, in a system wherein the co-culture of cells with different culture media is desired, fully or partially connected microchambers may be constructed by rows of microchambers being connected by shortening the cutter walls between them, such that compounds can diffuse between the compartments through the substrate. In the case where the skilled person will investigate individual layers of the substrate through forming partially connected microchambers, that is also within the scope of the invention. Of course, discrete microchambers that are fully isolated are also possible. In such cases, the cutter walls contact with the substrate platform. Therefore, in some embodiments, the cutting walls are configured to make discrete microchambers in the substrate. In some embodiments, the cutting walls are configured to contact the substrate platform.

[0062] In some embodiments, there is provided a method of making a multi-compartment 3D culture, the method comprising:

[0063] i. introducing one or more substrates into the mould by any method described herein, and ii. engaging the cutter as described herein to the mould comprising the substrate from step i.

[0064] Fluid Component

[0065] In some embodiments, there is provided a mould comprising a substrate platform, wherein the substrate platform is configured to retain one or more substrates, and wherein the mould is configured to receive a cutter comprising a plurality of cutting walls configured to cut a plurality of compartments into a substrate, wherein the cutter comprises a plurality of vessels, wherein each vessel comprises an inlet and an outlet, wherein the vessels are conduits to one or more of thecompartments. In some embodiments, there is provided a cutter implement as described herein, additionally comprising vessels configured as conduits away from one or more of the compartments. The vessels acting as conduits may be referred to as the “fluidic component” or “microfluidic component”. In some embodiments, there is provided a cutter implement as described herein, wherein one or more vessels are configured as conduits to one compartment and one or more different vessels are configured as conduits away from the same compartment. In some embodiments, there is provided a cutter implement as described herein, wherein one or more vessels are configured as conduits to more than one compartment and one or more different vessels are configured as conduits away from more than one compartments. In some embodiments, the cutting implement as described herein comprises a plurality of microfluidic channels. The cutter implement comprising the plurality of microfluidic channels may also be referred to as a “microfluidic device” or “microfluidic chip”. A “microfluidic chip” may also refer to the device additionally comprising the substrate. Therefore, it may also be described such that it is the microfluidic device that is configured to be a cutter, which forms the compartments as described herein. In this embodiment, it may be said that the device according to the invention comprises at least a mould assembly, as described herein, and a multi-chambered microfluidic device with integrated cutting implements. As would be clear to the skilled person, scalability of the fluidic component is within the scope of the invention, as are the various input and output combinations referred to herein.

[0066] When the cutting implement comprises fluidic or microfluidics channels the labour-intensive and imprecise process of moving gel samples into well-plates or open microfluidic compartments is avoided. Instead, the cutting of the samples simultaneously directly integrates and compartmentalizes them into the resulting microfluidic chip. It may be seen that the compartments or samples formed in the substrate by the cutter, may now be termed “chambers” or “microfluidic chambers” of the microfluidic device or chip, the terms are used interchangeably throughout. In instances when the device does not require moving the gel samples, even fragile tissues orTMMs may be incorporated. A further benefit when incorporating a microfluidic device, as described above, also is the avoidance of labour-intensive manual refreshing of cell culture medium. This also provides the user with the option of using only small volumes of culture medium and chemicals. It is therefore foreseen that a one-step production of a plurality of compartments or chambers in combination with the microfluidic system as described herein, allows for high throughput screening. This high throughput screening has particularly beneficial application in, for example the screening of drugs and / or therapies. In some embodiments, the microfluidic chip comprises 6 microchambers connected in parallel to a shared microfluidic inlet and outlet, forming one row. Therefore, in some embodiments, the microfluidic chip comprises 16 rows of 6 microchambers, or 24 rows of 6 microchambers, or 24 rows of 8 microchambers. In some embodiments, the microfluidic chip comprises 96 microchambers. In some embodiments, the microfluidic chip comprises 192 microchambers.In some embodiments, the microfluidic conduits are configured to be connected to leak-free connectors. For example, the microfluidic conduit outlet comprises Luer slip or Luer lock connectors, being a leak-free connection of fluidic tubing to any suitable pumps already. Alternative connectors and pumps will be known to the skilled person. In some embodiments, one or more microfluidic conduits are coupled to one syringe or pump. In some embodiments, one or more microfluidic conduits are coupled to a single syringe or pump containing at least 16, or at least 24 syringes. For example, one may incorporate 16 syringes (one per row along the short side) or 12 (one syringe per fluidic unit of 8 chambers in the 6x16 configuration) or 6 (one syringe per row on the long side). Such an embodiment is configured so that each row of microchambers receives a unique medium, or to a more complex system that e.g. automatically mixes stock solutions to create a variety of fluid streams with different concentrations of compounds of interest. In some embodiments, the fluid or microfluidic conduits are configured to receive medium from reservoirs on either side of the row of chambers or microchambers. In some embodiments, medium is manually delivered into the medium reservoirs. Such an embodiment beneficially avoids the need for pumps by using gravity for flow of medium.

[0067] In some embodiments, the rows of chambers are isolated from each other. In some embodiments, the rows of chambers are connected to each other. For example, in a system wherein the co-culture of cells with different culture media is desired, a connected system may be constructed. Rows of chambers may be connected by shortening the cutter walls between them, such that compounds can diffuse between the chambers through the substrate. Therefore, as previously discussed, in some embodiments, the cutting walls are configured to partially cut the substrate to form such chambers. Such an embodiment, may also be modified to allow media to perfuse though the substrate, such as by creating a pressure differential across the connected rows of chambers. As can be appreciated, the versatility and simplicity of the device of the invention makes it suitable for a wide-number of uses.

[0068] Once the tissue construct is prepared and / or inserted into the mould assembly, the cutter lid may be aligned and secured. The application of pressure may be used to facilitate precise slicing of the material into individual compartments while simultaneously incorporating the samples into the fluidic system. Therefore, in a further aspect, there is provided a method of making a microfluidic device, the method comprising:

[0069] i. introducing one or more substrates into the mould assembly as described herein.

[0070] ii. connecting the cutter comprising the fluidic component as described herein to the mould assembly of step i.

[0071] Microfluidic Setup

[0072] Sample chambers may be connected in groups in parallel to a common inlet and outlet, as described above. These groups of chambers may further be called “fluidic units”. The inlets and outlets may be compatible with luer slip connectors, allowing for direct attachment of tubing orattachment to a fluidic circuit board. For applications requiring differential culture conditions, fluidic units may be isolated or interconnected by adjusting the height of the cutter walls between chambers, enabling diffusion between adjacent compartments. Suitable adaptations and compatible fluidic circuit boards will be known to the skilled person and are within the scope of the invention. In one example, 8 sample microchambers (along the length of the slide) are connected in parallel to a shared fluidic inlet and outlet, forming a row covering half the length of the chip. In a further example, 12 such rows (2 along the length x6 along the width of the chip) cover the area of the chip.

[0073] Depending on the use case, each fluidic unit may be coupled to its own (syringe) pump, or to a single syringe pump containing 24 syringes so that each row gets a unique medium, or to a fluidic circuit board, or to a more complex system e.g. the chip may also be placed on a rocker table so that medium constantly flows back and forth. The drawback of this method is that it still requires frequent pipetting of medium, cannot reach high flow, and offers limited control overflow.

[0074] Fluid flow

[0075] The system is configured to facilitate fluid transport under controlled pressure differentials, thereby accommodating a range of viscosities and ensuring stable flow characteristics. For example, for a fluid exhibiting a dynamic viscosity of approximately 1 mPa s, the volumetric flow rate within the channel may be achieved up to approximately 80 mL / min, depending on the applied pressure differential. Specifically, under a pressure drop of 1 kPa, a flow rate of approximately 88pL / min is achievable, whereas an increased pressure differential of 100 kPa results in a flow rate of approximately 9 mL / min. The measurement of dynamic viscosity and pressures are known to the skilled person, and include the measurement by viscometers and / or rheometer for the viscosity measurements and pressure sensors for the pressure differential measurements.

[0076] The system may be adapted for biofluidic applications, wherein higher-viscosity fluids, such as hydrogel-based bio-inks with viscosities as high as 1 Pa s, may be utilized within the channel. The disclosed design facilitates precise control over flow-rates while mitigating excessive pressure buildup, thereby ensuring compatibility with shear-sensitive materials. Furthermore, the channel geometry is also suitable for the controlled flow of gases, wherein compressibility effects may be considered to optimize gas transport through the system.

[0077] In certain configurations, the dimensions and flow control mechanisms may be adjusted to achieve flow rates at the nanoliter scale, enabling precise handling of minute fluid volumes.

[0078] Analysis

[0079] In a further aspect, there is provided a method of screening drugs wherein,

[0080] i. the drugs are introduced to the microfluidic device as disclosed herein; and

[0081] ii. the drug interaction is measured and / or imaged.Therefore, in some embodiments, the device as described herein is used in a method for the screening or drugs. In some embodiments the device of the invention is used in biomedical research. Further applications include but are not limited to material degradation, food science, chemical analysis.

[0082] In some embodiments, the mould assembly comprises a polymer. For example, the walls of the cutter may puncture a small distance into the substrate platform, which may be covered by a thin polymer layer, thereby contributing to a water-tight seal between compartments. Alternatively, the device may include another separate sealing mechanism, such as an adhesive, and / or compression seal at the base of the compartments, to achieve a leak-prevention function without requiring direct puncturing of the polymer layer.

[0083] As discussed above, in some embodiments, the device of the invention is designed for compatibility with a standard microscopy slide. The dimensions of a standard microscope slide will be known to one of skill in the art, such as those typically 25x75 or 26x76 mm. In some embodiments, the device as disclosed herein is 5 to 15 mm thick. When the device of the invention is compatible with standard laboratory equipment, this may include automated scanning equipment, high throughput screening is further facilitated. Therefore, in one embodiment, the microfluidic device, optionally comprising an additional cover glass, is configured to allow imaging by a microscope and / or fluorescent screening equipment. Suitable microscopes and fluorescent screening equipment will be known to the skilled person to include, but not be limited to, Confocal microscopes, Light sheet microscopes, Raman microscopes, Widefield fluorescence microscopes, Phase-contrast microscopes, Brightfield microscopes, Multiphoton microscopes, High-content screening systems, Super-resolution microscopy systems, Optical coherence tomography (OCT) systems, and Automated imaging platforms.

[0084] A suitable thin cover slide will be known to one of skill in the art to include a standard microscope cover slide, or one suitable for the intended use, imaging or equipment, such as those being 18 mm to 24 mm, or rectangles with a width of 24 mm and a length of 32 to 60 mm. The thicknesses of cover slides may be 0.13 - 0.17 mm. Further optional configuration will also be within the scope of the skilled person. In one embodiment, the microscopy coverslip may be removably placed between an inner and outer mould. Such an embodiment allows the microscopy coverslip to be removable from the assembly to facilitate ease of extraction of samples for further analysis. In some embodiments, such an assembly may be fixed in place. For example, the microscopy coverslip may be glued in place on the outer mould. Such an embodiment of the device of the invention may be considered to be a simplified alternative by foregoing the need for an inner mould. Suitable adhesives and their quantities will be known to the skilled person. In some embodiments, the mould including the coverslip may be manufactured as a single component. The single component may then be configured for microscopic imaging.Further imaging agents may also be considered by the skilled person, such as any chemical or biological agent for perfusion through the microfluidics vessels. Such agents may include, but are not limited to, drug candidates, stimulation by light, electrical stimulation, such as through optional incorporated microelectrode arrays, or mechanical stimulation by optional pillars attached to a membrane on top of the substrate compartments, which may then be configured to be moved with air pressure. Therefore, possible compatible read-outs include at least microscopic imaging, fluorescent and colorimetric assays, assays on the perfused liquid from the microfluidics, and electrical signals detected by the optional microelectrode arrays.

[0085] Fabrication

[0086] One or more of the components of the device according to the invention can be fabricated using one or more of laser cutting, die cutting, milling, press cutting, layer-by-layer fabrication, 3D printing, or lithography. In a preferred embodiment, one or more of the components of the device according to the invention is fabricated using 3D printing. The devices according to the invention comprises one or more of, the mould assembly, a lid, the cutting implement, and further optional components to facilitate the use thereof. For example, should a water-tight seal be required, as discussed above, the device may further comprise further optional components, such as a connecting means which interlock the cutting implement and mould assembly. In some embodiments, one or more of the devices according to the invention are fabricated using 3D printing, plaster casting, moulding or a combination thereof.

[0087] In addition to the fabrication methods described above, the device or its components may be manufactured using a variety of methods, depending on the material properties, precision requirements, and scalability considerations. One such methods is Injection Molding for high-volume production of components with consistent dimensions and surface finish, particularly for polymer-based parts. This method allows for cost-effective mass manufacturing while maintaining structural integrity and reproducibility. Precision machining techniques such as milling, turning, and grinding may be utilized for fabricating components from rigid materials, including metals, ceramics, and engineering-grade polymers, ensuring tight tolerances and high mechanical strength.

[0088] When microfluidic features or elastomeric components are required, soft lithography techniques such as PDMS casting can be employed to create flexible, biocompatible, and optically transparent structures. This is particularly advantageous for applications involving biological samples or fluidic interactions.

[0089] Thermoplastic materials may be shaped using vacuum forming or thermoforming to produce thinwalled, lightweight, and durable components with complex geometries.In cases where porous or fibrous structures are required, electrospinning techniques may be employed to fabricate nanofiber networks that provide enhanced surface area, mechanical flexibility, and biointegration properties.

[0090] In addition to standard fused deposition modeling (FDM) 3D printing, Stereolithography (SLA) & Digital Light Processing (DLP) Printing methods may be used for the fabrication of high-resolution, fine-featured components, particularly where optical clarity or intricate structural details are required.

[0091] Components may be coated or functionalized using Chemical (CVD), or Physical Vapor Deposition (PVD), dip-coating, elevtrophoretic deposition (EPD) or sputtering techniques, enabling surface modifications such as hydrophobicity, antimicrobial properties, conductivity, or enhanced biocompatibility.

[0092] The selection of one or more of these fabrication methods allows for optimization of the device's mechanical, chemical, and biological properties, ensuring compatibility with the intended application while maintaining manufacturability and scalability and will be known to the skilled person.

[0093] In some embodiments there is provided a kit comprising the cutter as described herein, the mould as described herein and an instruction manual, or any combination thereof. In some embodiments there is provided a kit comprising the cutter as described herein, the mould as described herein and an instruction manual, or any combination thereof, and or more sealing elements, pre-filled reagent reservoirs, and / or tissue-mimicking materials.

[0094] Description of the figures

[0095] Figure 1. Sketch of the invention. A) A bottom view of the combined cutting implement I microfluidics system. The grid of sample compartments can be seen, as well as the walls used to cut the TMM samples. B) Top-view of the mould including outer mould, inner mould positioned on top ofthe glass below, and TMM layer. The dark circles represent holes for the screws. C) Front-view intersection ofthe invention when a layer of tissue mimicking material (TMM) is being cast. A stamp lid presses the TMM from one side to the other, with air and excess TMM exiting through a hole in the lid. The lid can be secured with four screws in the corners. Stamp lids are available in multiple thicknesses, so that different thicknesses of TMM layers can be cast. The TMM is cast on top of a PDMS-coated glass slide, and a ring called the "inner mould” makes sure that all TMMs are visible for microscopic imaging through the glass substrate and makes a water tight seal to the glass. This seal may be achieved by casting the whole inner mould out of PDMS or by attaching a layer of PDMS to the bottom of the inner mould. D) Front-view intersection of the TMM being cut into samples. E) Magnified look at an example of a multi-layer TMM. This TMM has three different layers. The first has no cells, the second has cells dispersed throughout, and the third has cells on the TMM layer interface. F) A magnified look at two sample compartments with parallel in- and outlets. Notice thatthe sharp walls of the cutter puncture a small distance into the layer of PDMS on the glass, which helps to prevent leakage between compartments. G) A magnified look at an alternative design for the sample compartments, illustrating how multiple rows of samples could be in contact by incorporating shorter cutter walls between rows with different culture media. Different cells can be seeded in the different rows and different cell culture media used. The cell culture media as well as signal molecules can diffuse between the compartments through the TMM. H) Alternative design for the mould where the glass cover slip is glued in place. I) Alternative design for the mould where the mould and microscopy cover slip are cast as a single piece of plastic.

[0096] Figure 2. Images of prototype (components of) device. A) The outer mould. B) An alternate design of the outer mould with a grid structure to support the glass cover slip. C) Outer mould from B including a glass cover slip and inner mould. D) A stamp lid. At the top the spillover slit can be seen. E) Four stamp lids of increasing thickness from left to right, for manufacture of (layered) tissue constructs of various thicknesses. F) Cutter lid: A multi-chambered lid with integrated cutting implements, seen from the underside so that the grid of chambers is visible. Each chamber has its own inlet and outlet holes in opposite corners of the chamber. G) An assembled device seen from above, showing the top of a transparent version of the cutter lid from F. At the bottom, two rows of six inlets and outlets are visible, and through the transparent cutter lid the fluidic channels between them can be seen. At the top, two fluidic connector pieces have been attached to the inlets and outlets. H) An open-top cutter lid seen from below, and I) from the side, and J) zoomed in on the cutter walls. K) An assembled device chip seen from above, using the same opaque cutter lid from F. It uses a rigid metal backbone, and has been screwed closed.

[0097] Figure 3. The device in use. A) Alginate gel deposited into one side of the mould. B) A stamp lid of known thickness is pressed onto the gel in the mould, spreading it out. The spillover slit is placed on the opposite side of the gel. C) Excess gel spills out of the spillover slit once the gel has been pressed through the whole mould. D) The sheet of alginate gel after removal of the stamp lid. E) A piece of cheese placed into the mould assembly as an example of an external sample. F) The cutter lid positioned to cut the piece of cheese into 96 samples. G) The piece of cheese cut into 96 samples and incorporated into individual sample chambers, seen from the bottom of the device. H) An alginate gel cut by an open-top cutter lid and incorporated into individual sample chambers, the samples visible through the holes of the open-top cutter. I) An alginate gel cut by a fluidic cutter lid and incorporated into separate sample chambers. The image shows a zoom-in on the bottom of the device, and the inlet and outlet holes in each chamber can be seen.

[0098] Figure 4. Analysis conducted on the device A) The device containing alginate samples under a fluorescent microscope. This particular microscope images from the top which is why the device is flipped upside down. Under an inverted microscope the device is imaged correct side up. B) The 96 alginate samples removed from the device, which uniquely allows for further analysis outside of the organ-on-chip platform. C) A microscopic image of those alginate samples removed from the device (1X objective). They are of a neat square shape, and the uninterrupted white lines betweenthem, where the cutter walls used to be, show that the cutter walls cut cleanly through the whole gel.

[0099] Figure 5. Fluidic channels and connections. A) A liquid containing a dye was pumped into channels on one half of the device assembly with the fluidic lid. B) A liquid containing a dye was pumped through the entire device assembly with the fluidic lid filling up the compartments.

[0100] Figure 6. Three different views of Open-Top device in which there are only chambers integrated to the cutter. The chambers are accessible from the top for adding any necessary liquids and compounds.

[0101] Figure 7. The device with fluidic channels integrated in three different views. The chambers top wall are designed in an angle so that the outlet of the chamber stays at the highest point of the chamber, allowing facile removal of any potential air bubbles.

[0102] Figure 8. Three different views of the mould of the device. Current design enables the insertion of different substrates to the bottom of the mould such as a glass cover slip for easy access to the samples using microscopy.

[0103] Figure 9. Inner mould of the device in three different views. This mould serves multiple functions, such as concentrating the samples into the viewpoint of the platform that enables access to all the samples from bottom by any indirect analytical techniques such as microscopy. In addition, this part aids the casting of liquid material into the zone on interest (i.e., where the cutters cutting part sits) of the platform. In addition, in the case of using fragile substrate such as a glass cover slip, this material cushions the contact of cutter and the mould and glass, preventing any mechanical damage to the glass.

[0104] Figure 10. The “stamp” lid in three different views. The lid serves multitude of functions such as enabling uniform thicknesses throughout the sample (in case of casting the samples) and / or enabling the formation of imprints on the sample surface. The lid can be made in multitude of thicknesses, shapes, etc.

[0105] Figure 11. A) Perspective view of “stamp” lid B) Top view of Open-Top cutter lid C) Perspective view of Open-Top cutter lid D) Perspective view of the mould E) Perspective view of the inner mould.Examples

[0106] Methods

[0107] Fabrication of device

[0108] Autodesk Inventor was used to design the device. After exporting the high-resolution stereolithography (STL) file, the platform was 3D printed using a commercially available digital light processing (DLP) printer and a biocompatible printing resin. After printing, the printed platform was washed in isopropanol for 30 minutes and cured under 60°C and 365 nm Ultraviolet (UV) light source for an hour. The platform was post-cured afterwards for one hour at 120°C to ensure biocompatibility.

[0109] Fabrication of tissue constructs

[0110] Tissue constructs can be either produced inside of the mould assembly (Fig. 3A-D), or prepared externally and placed into the device (Fig. 3E). To produce tissue construct inside the mould assembly, injectable or non-injectable tissue mimicking materials (TMMs) are used, optionally containing cells, spheroids, organoids, particles, or other additives. These materials are cast into the mould and shaped into uniform sheets of controlled thickness using an interchangeable lid system (Fig. 1C). The material is deposited in the mould on one side, and a stamp lid is pressed down into the mould such that the stamp lid presses the material to fill the whole mould and any excess material spills out of the slit in the stamp lid on the other side of the mould (Fig. 1C, 3A-C).

[0111] The stamp lids may be patterned to stamp specific structures into the TMM sheets. These patterns may be the same for each sample chamber, or they may be different to facilitate high-throughput production of a wide variety of patterned / shaped samples. To achieve multi-layered constructs, sequential casting is performed, with each layer containing different cell types or chemical additives as required (Fig. 1E). Combinations of the above are possible, such as placing a tissue slice in between two layers of biomaterials. Tissue interfaces can also be constructed this way.

[0112] External tissue construct such as porous scaffolds or tissue slices, including ex vivo biopsy samples, are directly placed onto the mould for subsequent processing (Fig. 3E-F). A cutter that only cuts along the outermost walls of the chip can be used to cut excess material from an external sample, making sure the sample fits exactly into the mould assembly. If the tissue slice is homogeneous this simply provides many compartmentalized samples for high throughput screening. If the tissue slice is heterogeneous in composition and / or structure, this can allow a “pixelated” scanning of the response of different areas of the culture slice such as different layers of skin or bone-cartilage interface. If patient biopsy samples are used for the tissue slices, this can be used for screening for personalized medicine.

[0113] Compartmentalization and Microfluidic Integration

[0114] Once the tissue construct is prepared and / or inserted into the mould assembly, the cutter lid is aligned and secured onto the material. The application of pressure facilitates precise slicing of thematerial into individual compartments while simultaneously incorporating the samples into the fluidic system (Fig. 3F-I).

[0115] Microfluidic Setup

[0116] Sample chambers are connected in groups in parallel to a common inlet and outlet (Fig. 1F, 2G). These groups are called fluidic units. The inlets and outlets are compatible with luerslip connectors, allowing for direct attachment of tubing or attachment to a fluidic circuit board. For applications requiring differential culture conditions, fluidic units are isolated or interconnected by adjusting the height of the cutter walls between chambers, enabling diffusion between adjacent compartments (Fig. 1G).

[0117] In one example, 8 sample microchambers (along the length of the slide) are connected in parallel to a shared fluidic inlet and outlet, forming a row covering half the length of the chip. 12 such rows (2 along the length x6 along the width of the chip) cover the area of the chip (Fig. 2F-G).

[0118] Depending on the use case, each fluidic unit can be coupled to its own (syringe) pump, or to a single syringe pump containing 24 syringes so that each row gets a unique medium, or to a fluidic circuit board, or to a more complex system that e.g. automatically mixes stock solutions to create a variety of fluid streams with different concentrations of compounds of interest.

[0119] Alternatively, the microfluidic channels instead may connect to medium reservoirs on either side of the row of microchambers. The need for pumps can be avoided by pipetting medium into the medium reservoirs and using gravity for flow of medium. The chip can be placed on a rocker table so that medium constantly flows back and forth. The drawback of this method is that it still requires frequent pipetting of medium, cannot reach high flow, and offers limited control overflow.

[0120] Example 1 Simple assay

[0121] The device is constructed and set up as detailed above.

[0122] Samples are stimulated (bio)chemically by pumping (or gravity-fed flow of) liquids containing the desired compounds through the sample chambers, mechanically by controlling the flow rates of pumped liquids, and optically by light through the cover slip. In some embodiments, mechanical stimulation is applied via flexible pillars or magnetically controlled implements. In some embodiments, electrical stimulation is applied via electrodes or micro-electrode arrays.

[0123] The device, being designed for compatibility with standard microscopes, is then used with automated imaging systems and fluorescent scanners (Fig. 4A). Assays or staining is performed by pumping the assay components through the sample chambers and subsequent imaging of samples through the cover slip. Alternatively, integrated sensors report real-time measurements of aspects such as pH, ionic strength or metabolite concentration are also monitored. Fluidic effluent from the microchannels is collected for biochemical assays, and in setups incorporatingmicroelectrode arrays, electrical readouts are obtained to assess electrophysiological activity. The device uniquely enables removal of samples from the fluidic system to perform any other analyses that are not possible to do on-chip (Fig. 4B-C).

[0124] Example 2 High-throughput Assay

[0125] The device is constructed and set up as detailed above. One chip currently contains 96 (8x12) square samples that are 3x3 mm in size and typically 0.25-1 mm thick. The design is easily altered to accommodate different shapes and numbers of samples if desired. Further miniaturization is possible, mainly limited by the thickness of the cutting walls. For example, a chip that contains 48 rectangular samples of 3x6 mm, 192 (8x24) square samples of 2x2 mm, or 384 (16x24) square samples of ~1.5x1.5 mm.

Claims

22Claims1. A cutter comprising a plurality of cutting walls configured to cut a plurality of compartments into a substrate,wherein the cutter comprises a plurality of vessels,wherein each vessel comprises an inlet and an outlet,wherein the vessels are conduits to one or more of the compartments.

2. A cutter according to claim 1 , additionally comprising vessels configured as conduits away from one or more of the compartments.

3. A cutter according to claim 2, wherein one or more vessels are configured as conduits to one compartment and one or more different vessels are configured as conduits away from the same compartment.

4. A cutter according to claim 2, wherein one or more vessels are configured as conduits to more than one compartment and one or more different vessels are configured as conduits away from more than one compartment.

5. A cutter according to any one of claims 1 to 4, wherein the vessels are microfluidic vessels.

6. A cutter according to any one of claims 1 to 5, wherein the cutting walls are configured to partially cut the substrate or wherein the cutting walls are configured to fully cut the substrate.

7. A cutter according to any one of claims 1 to 6, wherein the cutter is configured to cut at least 48 compartments, at least 96 compartments, or at least 384 compartments into the substrate.

8. A cutter according to any one of claims 1 to 7, wherein the substrate comprises one or more tissue mimicking materials.

9. A mould configured to receive the cutter according to any one of claims 1 to 8.

10. The mould according to claim 9, wherein the mould is configured to receive one or more substrates, wherein the substrate optionally comprises one or more tissue mimicking materials.

11. A cutter according to any one of claims 1 to 8, or a mould according to claim 9 or claim 10, wherein the cutter and / or mould comprises one or more materials selected from a 3D-printable polymer, a thermoplastic, a biocompatible polymer, a metal, a composite material, and a ceramic, more preferably the cutter and / or mould comprises one or more materials selected from polylactic acid (PLA), polyethylene terephthalate glycol (PETG), polycarbonate (PC), acrylonitrile butadienestyrene (ABS), polyether ether ketone (PEEK), silicone, hydrogels, stainless steel, titanium, aluminum, polydimethylsiloxane (PDMS), glass-reinforced composites, and ceramic-based materials.

12. A method of making a multi-compartment 3-D cell culture, the method comprising:i. introducing one or more substrates into the mould according to claim 9 or claim 10; ii. connecting the cutter according to claims 1 to 8 to the mould from step i.

13. The method according to claim 12, wherein the cutter is reversibly connected to the mould.

14. The method according to claim 12 or claim 13, wherein the cutter is connected to the mould with a water-tight seal.

15. A kit comprising the cutter according to claims 1 to 8, the mould according to claims 9 or claim 10 and an instruction manual.

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