Multiwell plates for creating high-density arrays of compartmentalized cell cultures for use in high-volume applications.

Abstract

The present disclosure relates to multi-well plates having fluid connections between adjacent wells that are useful for making cell culture substrates and conform to the American National Standards Institute's Laboratory Automation and Screening Society (ANSI / SLAS) microplate standard.

Description

Technical Field

[0001] The present disclosure relates to novel substrates for creating high-density arrays of compartmentalized cell cultures for use in rational drug discovery applications. Specifically, in some embodiments, the present disclosure relates to multi-well plates that comply with the Microplate Standards of the American National Standards Institute / Society for Laboratory Automation and Screening (ANSI / SLAS), in which groups of wells are fluidically connected to create cell culture substrates that can be used in a wide range of axonal transport assays in neurobiology research and drug discovery.

Background Art

[0002] Since being conceptualized in the mid-1970s, compartmentalized cell cultures (CCCs) have gained momentum as an important methodology in neurobiology research. Campenot, R.B., Proc. Natl. Acad. Sci. U.S.A. 74:4516-9 (1977). For example, CCCs can be used to study mechanisms such as synaptic transmission (Vikman et al., J. Neurosci. Methods 105:175-184 (2001)), axonal transport of proteins and organelles (Bousset et al., Ann. Neurol. 72:517-524 (2013)), or to study network communication between individual populations of neurons for the purpose of studying cell network formation (Taylor et al., J. Neurosci. 33:5584-5589 (2013)). Furthermore, CCCs have been used in experiments to study cell signaling between different cell types, such as neuron-muscle cell signaling (Zahavi et al., J. Cell Sci. 128:1241-1252 (2015)), or transmission between neurons from different brain regions (Berdichevsky, Y., Staley, K.J. & Yarmush, M.L. Lab Chip 10, 999-1004 (2010)). Traditionally, cell-chain complex (CCC) assays have been primarily used in basic research applications where the need for high-throughput or parallelization of experiments is limited. However, with the increasing demand from the pharmaceutical industry for more advanced, translation-relevant cell-based assays, the ability to use CCCs for drug screening applications is now required. For example, there is now great interest in gaining access to assay platforms that can model prions and prion-like mechanisms in relevant ways and enable the screening of thousands of compounds within relatively short timeframes (Zhang, M., Luo, G., Zhou, Y., Wang, S. & Zhong, Z. Phenotypic screens targeting neurodegenerative diseases. J. Biomol. Screen. 19, 1-16 (2014)). However, current state-of-the-art products cannot provide sufficient robustness or throughput to meet such demands.

[0003] To establish CCCs, cell culture substrates are used in which individual cell culture regions (wells) are fluidically interconnected via extremely small tubes with diameters large enough to establish fluid connections between wells, but small enough to prevent cells from migrating between different cell culture regions. Traditionally, CCCs were achieved by manual and very crude means, such as manually creating grooves or scratches in the bottom of cell culture dishes using a scalpel. The scratches were then sealed using vacuum grease, and then discontinuous regions were formed by carefully placing a physical barrier, such as a glass or polytetrafluoroethylene (PTFE) ring, over the sealed scratches. For a description of this method, see Campenot, RB, Proc. Natl. Acad. Sci. USA 74:4516-9 (1977). This method can be used to create substrates suitable for CCC formation, but it is very cumbersome and suffers from a high failure rate. In recent years, microfabrication methods have been used to create substrates for CCC formation. For example, using soft lithography and polydimethylsiloxane (PDMS, i.e., silicone rubber) casting, microfluidic devices that are highly uniform and far easier to handle than the original handmade substrates have been fabricated. See Taylor et al., Nat. Methods 2:599-605 (2005), and Neto et al., J. Neurosci. 36:11573-11584 (2016). However, due to the rather complex microchannel network required to enable the formation of CCCs, these microfluidic devices also have several drawbacks that prevent them from being used in efficient experiments. For example, these devices are difficult to fill with liquid, prone to bubble formation, difficult to modify the surface, cause strong stress to cell cultures, prevent free diffusion of gases and nutrients into cells, and are prone to detachment over time. To improve the cell culture-related problems associated with such microfluidic devices, the literature has reported on a simplified device design consisting of two large-sized (5×5 mm) continuous cell culture wells directly interconnected by small microfluidic channels.Similar to the microfluidic devices described above, this device was also fabricated in PDMS using soft lithography. Using this type of device, it was possible to culture certain types of neurons that would not survive in typical microfluidic substrates (Lu et al, J. Neurosci. Methods 209, 35-39 (2012)). While all the microfabricated devices described above significantly improve upon the original Campeont chamber design, they clearly suffer from several drawbacks. In addition to the problems already mentioned, the large wells and complex microchannel layouts required for these substrates make it impossible to scale up these designs to the high-density formats necessary for high-volume screening. Furthermore, these microfabricated substrates are typically fabricated from soft polymer materials such as PDMS, which exhibit flexible and elastic material properties, making such materials unsuitable for fabricating devices requiring tight spatial tolerances, such as shielded microtiter plates. Finally, these microfabricated devices do not provide the ideal optical environment for the type of high-resolution imaging required to monitor axonal transport. Typically, axonal transport analysis requires imaging of neurites (cellular processes) located within fluid connections to measure the linear movement of target proteins and organelles. Therefore, it is essential that the fluid connections do not introduce differences in optical conditions such as variable contrast, refractive index, or light emissivity or transparency. For automated imaging, the fluid connections must be positioned in a coordinate accessible by the imaging instrument; i.e., fluid connections located on the wall between two wells are often inaccessible to automated imaging instruments. In summary, there is a need for designs and fabrication methods that enable the creation of microfluidic substrates in the form of ANSI / slas-compliant microtiter plates capable of providing high-density arrays of CCCs for drug screening applications. [Overview of the project] [Means for solving the problem]

[0004] Here, the inventors present a novel substrate that enables the formation of high-density arrays of CCCs, allowing for high-volume experimental applications such as medium-throughput screening and high-throughput screening (MTS and HTS, respectively). The substrate is based on, but not limited to, a standard 384-well plate format, in which at least two wells in the plate are interconnected by fluid connections in the form of closed channels that can be small enough to prevent cell migration and further maintain chemical integrity between wells, and such channels are essentially located in the plane of the bottom of the wells. Thus, the microtiter plate includes open wells and has an optically transparent bottom, and its surface properties allow for cell culture. The fluid connections are carefully designed to allow for robust fluid handling to ensure a high success rate in experiments, and the substrate can be easily surface-modified using wet chemical methods. Furthermore, the substrate is designed to provide optimal conditions for high-resolution imaging through careful selection of substrate materials, as well as the design and placement of the fluid connections. To enable use in HTS applications, the substrate is designed to conform to all ANSI / SLAS microplate standards and is therefore compatible with most commercially available fluid handling robots and optical readout systems.

[0005] According to this specification, the disclosure includes, for example, a multiwell plate containing wells, wherein at least two adjacent wells in the plate have at least one fluid connection through a wall separating the at least two adjacent wells, and the fluid connection is essentially located in the plane of the bottom of the wells. In some embodiments, the disclosure includes, for example, a multiwell plate containing wells, wherein at least two adjacent wells in the plate have at least one fluid connection, and the fluid connection partially traverses at least one well in the multiwell plate. In some embodiments, the disclosure includes, for example, a multiwell plate containing wells, wherein at least two non-adjacent wells in the plate have at least one fluid connection, and the fluid connection completely traverses an adjacent well to at least one of the non-adjacent wells, and the adjacent well also has an optical detection marker in the same plane as the at least one fluid connection to allow a microscope or high-content imaging system to autofocus in that plane. In some embodiments, the multiwell plate conforms to the American National Standards Institute Laboratory Automation and Screening Society (ANSI / SLAS) microplate standard. In some embodiments, the multiwell plate includes a substrate made from a thermoplastic material to facilitate high-precision manufacturing. In some embodiments, the thermoplastic material includes polystyrene (PS), cycloolefin copolymer (COC), cycloolefin polymer (COP), poly(methyl methacrylate (PMMA), polycarbonate (PC), polyethylene (PE), polyethylene terephthalate (PET), polyamide (Nylon®), polypropylene or polyetheretherketone (PEEK), Teflon®, PDMS, and / or thermosetting polyester (TPE). In some embodiments, the multiwell plate includes a substrate made from cycloolefin copolymer (COP), cycloolefin polymer (COC), or polystyrene (PS). In other embodiments, the multiwell plate includes a substrate made from silicon, glass, ceramic material, or alumina.In some embodiments, the plate comprises a substrate having two or more layers, which may be joined by ultrasonic welding, thermocompression bonding, plasma bonding, solvent-assisted bonding, laser-assisted bonding, or adhesive bonding using adhesive or double-adhesive tape. In some embodiments, the plate comprises a substrate coated with a protein or polymer. In some cases, the plate comprises a substrate coated with one or more of the following: poly-l-lysine, poly-L-ornithine, collagen, laminin, Matrigel®, or bovine serum albumin. In some cases, the plate includes a substrate having a surface chemically modified with one or more of the following: poly[carboxybetaine methacrylate] (PCBMA), poly[[2-methacryloyloxy]ethyl]trimethylammonium chloride] (PMETAC), poly[poly(ethylene glycol)methyl ether methacrylate] (PPEGMA), poly[2-hydroxyethyl methacrylate] (PHEMA), poly[3-sulfopropyl methacrylate] (PSPMA), and poly[2-(methacryloyloxy)ethyldimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH).

[0006] In some embodiments, the plate includes groups of at least 32 or at least 96 fluid-connected wells. In some embodiments, at least one fluid connection includes cross-sectional dimensions of at least 1 to 10 μm and up to 10 to 20 μm, and optionally has an aspect ratio in the range of 1:5 to 2:1 (height:width). In some embodiments, at least one fluid connection includes cross-sectional dimensions (H and / or W) of 1 to 20 μm, such as 1 to 5 μm, 5 to 10 μm, 10 to 15 μm, 15 to 20 μm, 5 to 15 μm, or cross-sectional dimensions (H and / or W) of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, or 20 μm, and may also have an aspect ratio (H × W) in the range of 1:5 to 2:1. In some embodiments, the fluid connection includes cross-sectional dimensions of 5 × 5 μm or less, or 3 × 3 μm to 5 × 5 μm. In some embodiments, the dimensions, shape, and number of fluid connections vary over the length of at least one fluid connection to improve nerve process penetration and productivity. In some embodiments, the length of at least one fluid connection is at least 0.5 mm and up to 5.0 mm. In some embodiments, the aspect ratio of the dimensions of at least one fluid connection ranges from 20:1 (W:H) to 1:5 (W:H).

[0007] In some embodiments, the multiwell plate includes 96, 384, 1536, or 3456 well formats, and is optionally organized into a 2:3 rectangular matrix. In some embodiments, the multiwell plate includes at least two groups of three adjacent fluidically interconnected wells. In some embodiments, the multiwell plate includes at least three groups of two adjacent fluidly interconnected wells. In some embodiments, the multiwell plate includes at least one group of four adjacent fluidly interconnected wells.

[0008] This disclosure also encompasses a method for high-throughput screening of a material of interest, which includes screening the material of interest using a multiwell plate described in any one of the embodiments herein. In some embodiments, the material of interest is a 2D cell culture. In other embodiments, the material of interest is a 3D cell culture.

[0009] Additional objectives and benefits are partially described in the following description, partially evident from that description, or can be learned through practice. These objectives and benefits are realized and achieved by the elements and combinations specifically indicated in the attached claims.

[0010] Please understand that both the general explanation above and the detailed explanation below are illustrative and descriptive only, and do not limit the scope of the claims.

[0011] The accompanying drawings incorporated herein and constituting part of this specification illustrate one (or more) embodiments and, together with the description, help illustrate the principles described herein. [Brief explanation of the drawing]

[0012] [Figure 1] This figure illustrates one method of manufacturing and assembling a microplate. Two layers (layers 2 and 3) are bonded together to form a laminate for sealing and defining fluid connections. This figure shows a combined laminate (layers 2 and 3) bonded to a bottomless 384-well plate (layer 1). Layer 2: A thick +1 mm substrate with milled through-holes matching the 384-well plate pattern, including deep grooves on the underside. Layer 1: A standard bottomless 384-well plate. Bottom (layer 3): A thin, transparent, preferably HCA-compatible sheet.

[0013] [Figure 2] This figure shows a substrate including connections between wells and illustrates two possible fluid connections between those wells.

[0014] [Figure 3] This diagram shows how different designs, namely paired wells, three-connected wells, and four-connected wells, can be packed in microplate format.

[0015] [Figure 4A] This is a side view of the substrate and a diagram showing how it can be assembled in a three-layer design. Layer 1: Standard top from a 384-well microtiter plate, Layer 2: Thick +1mm substrate with milled through-holes matching the 384-well plate pattern, including deep grooves on the bottom, Layer 3: Plate bottom, i.e., a thin film (100-200 μm) bonded to the upper Layer 2. [Figure 4B] This is a side view of the substrate, illustrating how it can be assembled in a two-layer design. Layer 1 is a standard top layer from a 384-well microtiter plate, and Layer 3 is the bottom layer of the plate, i.e., a thin film (100-200 μm) bonded to the upper Layer 2.

[0016] [Figure 5] This figure shows a substrate specifically designed for real-time study of axonal transport processes using a high-resolution imaging system. In this design, two non-adjacent wells are fluid-connected, with the fluid connection traversing the well located between the fluid-connected wells. This design requires a three-layer substrate.

[0017] [Figure 6] This figure shows a substrate specifically designed for real-time study of axonal transport using a high-resolution imaging system. In this design, two adjacent wells are fluidically connected, and the fluid connection can partially traverse one well to facilitate imaging within the fluid connection at a position that provides ideal optical conditions. This design requires a three-layer substrate.

[0018] [Figure 7]A diagram showing the concept of a prion progression and regulation assay. A prion-like mechanism inducer (e.g., pathogenic tau) can be added to well 1, and then the progression of pathogenesis can be regulated in well 2 and detected in well 3.

[0019] [Figure 8] A diagram showing a microscopic image of the spread of fluorescently labeled NDAP (tau particles) between cell cultures in adjacent wells connected by a fluid connection. The graph further demonstrates that the diffusion depends on the number of fluid connections and that cells are required for transport between wells.

[0020] [Figure 9] A diagram showing data from an experiment examining the retrograde axonal transport of α-synuclein oligomers.

[0021] [Figure 10] A diagram showing the concept for assaying axonal transport in primary neurons using the dynamic imaging and specialized substrates outlined in FIG. 6.

Best Mode for Carrying Out the Invention

[0022] The present disclosure relates to a novel substrate for generating a high-density array of compartmentalized cell cultures (hereinafter referred to as CCC) for use in high-volume applications such as medium-throughput screening and high-throughput screening (hereinafter referred to as MTS and HTS, respectively). Specifically, the present disclosure relates to a multi-well plate compliant with ANSI / SLAS standards, which may be a 384-well plate in some embodiments, wherein groups of wells are fluidly connected via microfabricated closed channels that are small enough to prevent movement of cells or cell clusters and / or to maintain chemical integrity between wells.

[0023] Definitions As used herein, the term “about” refers to numerical values, including, for example, integers, fractions, and percentages, whether expressly indicated or not. Generally, the term “about” refers to a range of numerical values ​​(e.g., + / - 5 to 10% of the listed range) that a person skilled in the art would consider equivalent to (e.g., having the same function or result as) the listed values. Where terms such as “at least” and “about” precede a list of numerical values ​​or ranges, those terms qualify all of the values ​​or ranges provided in the list. In some cases, the term “about” may include numerical values ​​rounded to the nearest significant digit.

[0024] As used herein, the term "or" should be interpreted as "and / or" unless the context makes it clear that only substitutes are intended.

[0025] A "multiwell plate" refers to a plate having wells or compartments that can be used as small test tubes. In some examples, a multiwell plate may have 96, 384, 1536, or 3456 wells arranged in a 2:3 rectangular matrix. The "substrate" of the plate refers to the general material that forms the plate structure and the wells of the plate. The substrate may include one or more layers and one or more coatings.

[0026] The term "384-well format" refers to a multi-well plate having 384 wells, i.e., 16 x 24 wells, arranged in a 2:3 rectangular matrix. In this context, the term "format" simply refers to the way the columns of wells are arranged (e.g., 2:2, 2:3, and the number of wells in each row that gives the total number of wells). Higher multi-well plate formats with 32 x 48 wells (1536 wells) or lower multi-well plate formats with 8 x 12 wells (96 wells) can also be used. The assumed plate formats may be, for example, 96, 384, 1536, or 3456 wells.

[0027] The terms “connected wells,” “interconnected wells,” or “fluid-connected wells” refer to wells that have direct fluid connections between them. “Neighboring wells” refer to adjacent wells, which may be interconnected by one or more fluid connections, forming a so-called “group” of wells. As used herein, a “group” of wells refers to wells that are directly or indirectly connected by fluid connections. In some embodiments, such groups of wells can form an “assayable structure,” “assayable entity,” or “assayable group,” i.e., a structure or entity used in an intended assay. For example, groups of at least three interconnected wells can form an assayable entity. Such groups of wells can be addressed individually, in parallel, or sequentially in multiple groups on a 384-well plate.

[0028] The term “fluid connection” between wells refers to a well having one or more connections or conduits that can enable controlled transport or prevention of transport of materials as intended. In this embodiment, the fluid connection consists of one or more microfabricated closed channels connecting two wells in a multiwell plate. In one embodiment, the fluid connection enables the growth / transport of axons and / or dendrites but prevents the transport of cells or cell bodies. In this embodiment, small molecules, polymers, proteins, and nanoparticles can be transported through the fluid connection between wells, and such transport can be regulated by manipulating hydrostatic pressure. In another embodiment, the fluid connection enables the transport of cells, cell clusters, and axons and dendrites. The term “cross-sectional dimensions” refers to the width and height of the fluid connection between two wells.

[0029] The "ANSI / SLAS Microplate Standards" refer to a set of standards that outline the physical dimensions and tolerances of footprint dimensions, height dimensions, bottom flange outer dimensions, well positions, and well bottom heights. For example, in some embodiments, a multiwell plate conforms to a valid ANSI / SLAS standard, namely ANSI / SLAS 1-2004(R2012): footprint dimensions, ANSI / SLAS 2-2004(R2012): height dimensions, ANSI / SLAS 3-2004(R2012): bottom flange outer dimensions, ANSI / SLAS 4-2004(R2012): well positions, and / or ANSI / SLAS 6-2012: well bottom height.

[0030] The term "thermoplastic material" refers to plastic materials, most commonly polymer materials, that become moldable or flexible above a certain temperature and solidify when cooled.

[0031] Compositions and methods A. Base material properties To meet the current requirements of commercially available medium-throughput and high-throughput screening (MTS-HTS) systems, the substrates of this disclosure may, in some embodiments, have a physical footprint and shape as specified in the ANSI / SLAS microplate standards. By adhering to these standards, embodiments of this disclosure may be compatible with established robotic plate handling systems, liquid handling systems, and optical readout systems used in MTS-HTS. However, since microplate standards are subject to future changes in shape or design, the present invention also adapts to multi-well plates of various shapes and sizes.

[0032] In one embodiment of the present disclosure, the substrate is composed of three parts.

[0033] Firstly, the substrate consists of a top portion that defines the external dimensions and shape of the substrate. The first portion also defines the macroscopic portion of the wells or cell culture area. The size and geometric shape of these wells should be designed to facilitate the liquid handling and cell culture processes. In one embodiment of this disclosure, a 384-well format is used. However, in other embodiments of this disclosure, multi-well plates having 96, 1536, or 3456 wells may be used (Figure 1). For example, in embodiments using a 384-well plate, the plate includes at least 96 groups of three adjacent fluidically interconnected wells, at least 192 groups of two adjacent fluidly interconnected wells, or at least 96 groups of four adjacent fluidly interconnected wells. Multi-well plates having 96, 1536, or 3456 wells, each having two or three interconnected well groups, may also be used.

[0034] The second and intermediate portions of the substrate define the fluid connections between wells (Figure 1). Depending on the application, the size and length of these fluid connections can be varied and depend on the type of cell-based assay in which the substrate is used. These connections are small enough to prevent cell migration between different cell culture zones (wells), for example, for assaying axonal transport mechanisms, such as prion-like diffusion of neurodegenerative disease-related peptides (Figure 2). In this case, the cross-sectional dimensions of the fluid connection section may be 1 to 20 μm, such as 1 to 5 μm, 1 to 10 μm, 5 to 10 μm, 10 to 20 μm, 10 to 15 μm, 15 to 20 μm, or 5 to 15 μm, or may include one or more connection sections having dimensions (H or W) of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, or 20 μm, and may also have an aspect ratio (HxW) in the range of 1:5 to 2:1.

[0035] Furthermore, to assay mechanisms related to axonal transport, a multiwell plate can be designed such that two non-adjacent wells have at least one fluid connection, and this fluid connection crosses a well adjacent to at least one of the non-adjacent wells, forming a detection zone, where optical conditions are optimized for high-resolution experiments using high-content imaging equipment (Figures 4 and 5). To facilitate autofocusing of the high-content imaging system, the wells may have optical detection markers in the same plane as at least one fluid connection. The cross-sectional dimensions of the fluid connection in this case may be between 1 and 20 μm, such as 1 to 5 μm, 1 to 10 μm, 5 to 10 μm, 10 to 20 μm, 10 to 15 μm, 15 to 20 μm, or 5 to 15 μm, or may include one or more connections having dimensions (H or W) of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, or 20 μm, and may also have an aspect ratio (HxW) in the range of 1:5 to 2:1. In some embodiments, the length of the fluid connection spanning one adjacent well is at least 3 to 5 mm.

[0036] In some embodiments, the cross-sectional dimensions of the fluid connection include less than 1 × 0.5 mm. In one embodiment, the fluid connection consists of one or more connection sections having small dimensions of approximately 3 × 3 μm. The connection sections can also have larger dimensions of up to 100 × 100 μm. The aspect ratio, i.e., the ratio of width to height of the cross-sectional dimensions, can be in the range of 20:1(W:H) to 1:5(W:H), such as 20:1 to 10:1, 10:1 to 5:1, 5:1 to 1:1, 2:1 to 1:2, 1:1 to 1:2, 1:1 to 1:5, or 1:2 to 1:5 (total W:H).

[0037] The shape and size of the fluid connector may vary along the long axis of the connector to optimize parameters such as productivity, fluid wetting and filling, and entry of cellular processes into the fluid connector. For example, incorporating a funnel-shaped structure at the inlet of the fluid connector can improve neurite guidance and penetration, and varying the height of the fluid connector can improve mechanical stability and therefore productivity. In one embodiment, a channel with dimensions of 6 × 8 μm (W × H) is expanded to 20 × 8 μm (W × H) over a distance of 200 μm, thereby improving the guidance of axons and dendrites into the fluid connector. In another embodiment, these funnel-shaped structures are joined together to form one large fluid connector at the inlet, further improving neurite guidance and penetration. In one embodiment, this large fluid connector at the inlet is also made higher, significantly improving the production yield of multiwell plates. In one embodiment, the height of the fluid connector is increased from 8 μm to 50 μm, but other heights are also conceivable.

[0038] Furthermore, the number of connected wells may vary depending on the assay application. In one embodiment of the present disclosure, the substrate comprises several units of paired wells (i.e., two connected walls); in a second embodiment of the present disclosure, the substrate comprises several units of three connected wells; and in a third embodiment of the present disclosure, the substrate comprises several units of four or more connected wells (Figure 3). In one embodiment of the present disclosure, the fluid connection is formed directly in the first layer of the substrate, thus completely eliminating the need to include a second layer within the substrate (Figure 4).

[0039] A third portion of the substrate defines the bottom of the substrate. To enable high-resolution imaging readout, in some embodiments, this bottom of the substrate is optically transparent in the visible and far-ultraviolet light spectral range and thin enough to allow imaging using a high numerical aperture microscope objective lens. Thus, in some embodiments, the thickness of the third bottom is less than 200 μm, such as 10–50 μm, 50–100 μm, or 100–200 μm. In other embodiments of the present disclosure where high-resolution imaging is not utilized, the bottom layer of the substrate can be made thicker to increase the mechanical robustness of the substrate (Figure 1). Therefore, in some embodiments, the thickness of the third bottom is in the range of 200 to 1000 μm, such as 200 to 500 μm, or 300 to 700 μm, or 500 to 1000 μm, or 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1000 μm.

[0040] In some embodiments, wells within the substrate may be equipped with optical markers located in the same plane as the fluid connection, enabling microscopes and high-content imaging equipment to autofocus and subsequently locate the fluid connection. Non-limiting examples of such optical markers are contrast-enhanced prints or contrast-enhanced microstructures.

[0041] B. Method for preparing the base material The substrates of this disclosure can be made from a wide range of materials, such as thermoplastic resins. Examples of thermoplastic materials include, for example, polystyrene (PS), cycloolefin copolymer (COC) or cycloolefin polymer (COP), poly(methyl methacrylate (PMMA), polycarbonate (PC), polyethylene (PE), polyethylene terephthalate (PET), polyamide (Nylon®), polypropylene, or polyetheretherketone (PEEK). Additional materials include perfluorinated materials such as Teflon®, silicone polymers such as PDMS, thermosetting polymers such as thermosetting polyester (TPE), or hard crystalline or amorphous materials such as ceramics like silicon, glass, or alumina. However, disposable substrates are preferred, and to meet the cost criteria for high-throughput screening where large quantities of substrate may be consumed, the substrates can be made from PS, COC, or COP because these materials are suitable for cost-effective mass production such as injection molding, hot embossing, or computer-aided manufacturing (CAM) micromachining. This is because it may be suitable for the method. In some embodiments, the material used for the substrate is suitable for a surface coating that enables cell culture. For example, in some embodiments, it may be desirable to perform physical surface treatment, such as plasma treatment or corona discharge, and to coat the substrate with material protein or polymer materials such as poly-l-lysine, poly-L-ornithine, collagen, laminin, Matrigel®, bovine serum albumin, or other protein solutions. Furthermore, chemical modifications such as poly[carboxybetaine methacrylate] (PCBMA), poly[[2-methacryloyloxy]ethyl]trimethylammonium chloride] (PMETAC), poly[poly(ethylene glycol)methyl ether methacrylate] (PPEGMA), poly[2-hydroxyethyl methacrylate] (PHEMA), poly[3-sulfopropyl methacrylate] (PSPMA), and poly[2-(methacryloyloxy)ethyldimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH) may also be grafted onto the surface.

[0042] Various joining methods can be used to assemble different layers of the substrate. For example, methods such as ultrasonic welding, thermocompression bonding, plasma bonding, solvent-assisted bonding, laser-assisted bonding, or adhesive bonding using adhesive or double-layer adhesive tape can be used. Although undesirable from a manufacturing standpoint, the substrate may be composed of different materials. In one embodiment of this disclosure, the bottom layer is composed of glass, and the other layers are composed of thermoplastic resin or silicone polymer material. Therefore, the embodiments disclosed herein are as follows: (1) A multiwell plate having an optically transparent bottom and having surface properties that enable cell culture, wherein at least two wells in the plate are directly fluid-connected via at least one closed microchannel of a sufficiently small size to prevent cell migration and minimize the diffusion of cells and biomolecules between wells, and the at least one microchannel is essentially located in the plane of the bottom of the well. (2) The multiwell plate according to (1), wherein a fluid connection is formed between at least two adjacent wells. (3) The multiwell plate according to (1), wherein a fluid connection is formed between at least two non-adjacent wells, and the fluid connection traverses the bottom of a well adjacent to at least one of the fluid-connected wells. (4) The multiwell plate according to (3), wherein the plate further includes an optical detection marker in at least one well that is fluidly connected to another well. (5) The multiwell plate described in (1), wherein the multiwell plate conforms to the American National Standards Institute's Laboratory Automation and Screening Society (ANSI / SLAS) microplate standard. (6) The multiwell plate according to any one of embodiments (1) to (5), wherein the multiwell plate includes a substrate made from a thermoplastic material. (7) The multiwell plate according to (6), wherein the thermoplastic material comprises polystyrene (PS), cycloolefin copolymer (COC), cycloolefin polymer (COP), poly(methyl methacrylate (PMMA), polycarbonate (PC), polyethylene (PE), polyethylene terephthalate (PET), polyamide (Nylon®), polypropylene or polyether ether ketone (PEEK), Teflon®, PDMS, and / or thermosetting polyester (TPE). (8) The multiwell plate according to any one of embodiments (1) to (7), wherein the multiwell plate comprises a substrate made from a cycloolefin copolymer (COP), a cycloolefin polymer (COC), or polystyrene (PS). (9) The multiwell plate according to any one of embodiments (1) to (8), wherein the multiwell plate includes a substrate made of silicon, glass, ceramic material, or alumina. (10) A multiwell plate according to any one of embodiments (1) to (9), wherein the plate comprises a substrate having two or more layers, and the layers are optionally joined by ultrasonic welding, thermocompression bonding, plasma bonding, solvent-assisted bonding, laser-assisted bonding, or adhesive bonding using an adhesive or double-adhesive tape. (11) A multiwell plate according to any one of embodiments (1) to (10), wherein the plate comprises a substrate coated with a protein or polymer. (12) The multiwell plate according to (11), wherein the plate comprises a substrate coated with one or more of the following: poly-l-lysine, poly-L-ornithine, collagen, laminin, Matrigel®, or bovine serum albumin. (13) A multiwell plate according to any one of Embodiments (1) to (12), wherein the plate comprises a substrate having a surface chemically modified with one or more of the following: poly[carboxybetaine methacrylate] (PCBMA), poly[[2-methacryloyloxy]ethyl]trimethylammonium chloride] (PMETAC), poly[poly(ethylene glycol)methyl ether methacrylate] (PPEGMA), poly[2-hydroxyethyl methacrylate] (PHEMA), poly[3-sulfopropyl methacrylate] (PSPMA), and poly[2-(methacryloyloxy)ethyldimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH). (14) A multiwell plate according to any one of embodiments (1) to (13), wherein the plate comprises groups of at least two, at least four, at least eight, at least sixteen, at least 32, or at least 96 fluid-connected wells. (15) A multiwell plate according to any one of embodiments (1) to (14), wherein at least one fluid connection portion includes a cross-sectional dimension (H and / or W) of 1 to 20 μm, such as 1 to 5 μm, 1 to 10 μm, 5 to 10 μm, 10 to 20 μm, 10 to 15 μm, 15 to 20 μm, or 5 to 15 μm, or includes a cross-sectional dimension (H and / or W) of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, or 20 μm, and optionally also has an aspect ratio (H × W) in the range of 1:5 to 2:1. (16) A multiwell plate according to any one of embodiments (1) to (15), wherein the fluid connection portion includes a cross-sectional dimension of 5 × 5 μm or less, or 3 × 3 μm to 5 × 5 μm. (17) The multiwell plate according to (16), wherein the dimensions, shape and number of fluid connections vary over the length of at least one fluid connection to improve nerve process penetration and productivity. (18) A multiwell plate according to any one embodiment (1) to (17), wherein the length of at least one fluid connection is at least 0.25 mm and at most 5.0 mm. (19) A multiwell plate according to any one of embodiments (1) to (18), wherein the aspect ratio of the dimensions of at least one fluid connection is in the range of 20:1 (W:H) to 1:5 (W:H). (20) A multiwell plate according to any one of embodiments (1) to (19), wherein the multiwell plate includes a 96, 384, 1536, or 3456 well format and is optionally organized into a 2:3 rectangular matrix. (21) The multiwell plate according to any one embodiment (1) to (20), wherein the multiwell plate includes at least two groups of three adjacent, fluidly interconnected wells. (22) The multiwell plate according to any one embodiment (1) to (21), wherein the multiwell plate includes at least three groups of two adjacent fluidically interconnected wells. (23) A multiwell plate according to any one embodiment (1) to (22), wherein the multiwell plate includes at least one group of four adjacent, fluidly interconnected wells. (24) A method for high-throughput screening of a material of interest, comprising screening the material of interest using a multiwell plate as described in any one of the above paragraphs (1) to (23). (25) The method according to (24), wherein the material of interest is a 2D cell culture. (26) The method according to (24), wherein the material of interest is a 3D cell culture.

[0043] example Example 1 - Assay for studying the diffusion of neurodegenerative disease-associated peptides (NDAPs) in neural circuits. The diffusion of neurodegenerative disease-associated peptides (NDAPs) into the brain is considered one of the major pathological mechanisms in progressive neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In this concept, the pathologically soluble forms of NDAPs, such as amyloid-β, α-synuclein, and tau proteins, are incorporated by neurons, leading to the progression of protein misfolding, synaptic decongestion, and neuronal cell loss. Furthermore, numerous studies have reported prion-like mechanisms of intracellular NDAPs, namely the intracellular transport of NDAPs and their diffusion from one neuron to another. Since neurodegenerative diseases remain virtually untreatable, a high-throughput assay platform that reflects all these complex neuropathological features in vitro and enables screening and profiling of a larger set of compounds to prevent this neurodegenerative cascade represents an urgent unmet medical need.

[0044] Using the substrates of this disclosure, the inventors were able to create unique high-throughput in vitro assays that reflect all the features of the neurodegenerative disease cascade within the CNS neural circuit. Mouse cerebral cortical neuron cultures were used because the neurons in these cultures develop a wide range of processes and form functional synaptic connections in vitro.

[0045] In detail, mouse cortical E18 neurons were plated onto a customized CCC substrate having a 384-well plate format containing 96 experimental units consisting of three adjacent wells fluidly connected. In this application, it was of paramount importance that cells could not move between adjacent wells; therefore, the fluid connections were smaller than the neuron cell bodies. Thus, each fluid connection consisted of 10 to 30 holes with a cross-sectional diameter of 6 × 8 μm. Before seeding the cells, the substrate was first coated overnight at 37°C with a 0.01% poly-L-ornithine solution. Subsequently, Ca 2+ / Mg 2+ Wash the wells with PBS containing Ca 2+ / Mg 2+Laminin diluted to 10 μg / ml was added in PBS containing [specific compound] and incubated at 37°C for 2 hours. Laminin was removed immediately before cell seeding. Cells were then prepared and cultured as described in Example 1. After 7 days of culture, cells in Zone 1 of all experimental units in the plate were treated with a 50 nM NDAP polymer solution, or patient-derived material such as pathogenic tau protein oligomers extracted from CSF of Alzheimer's patients. The plate was then returned to the incubator and the cells were cultured for a further 7 days. After 14 days of in vitro culture, the cells in the plate were fixed and stained for neurons and assay-specific markers using an immunocytochemistry protocol, and high-content imaging was used to describe synaptic NDAP uptake, intraneuronal diffusion and NDAP-mediated changes, and neuronal survival. Briefly, cells were fixed using PBS or 4% PFA in methanol. Neurons were evaluated using antibodies that bind to mouse MAP-2AB (1:1000), chicken MAP-2AB (1:10000), or bTubIII (1:1000). Hoechst (nuclear) staining was also performed. Anti-bTubIII (Sigma-Aldrich Sweden AB, Stockholm, Sweden) (1:1000), anti-PSD-95 (1:1000), anti-synaptophysin (1:1000), and anti-tau (1:1000) antibodies were combined with MAP-2AB antibody (Sigma-Aldrich Sweden AB, Stockholm, Sweden). High-content imaging (HCA) analysis was performed at 10x, 20x, or 40x magnification (PerkinElmer) using an Operetta® high-content imager.

[0046] To screen for modulators of NDAP diffusion across synaptic neurons, chemical, biological, or genetic interventions can be performed in well 2 of the experimental unit to modulate the cell culture capacity for NDAP diffusion, and well 3 of the unit is used to measure the intracellular presence of NDAP diffused from well 1 through the cell culture. A diagram illustrating the concept of the assay is shown in Figure 7. Using this assay concept, the inventors demonstrated NDAP uptake and regulation. After 7 days of in vitro culture, pathological tau extracted from CSF of human AD patients was added to the cells in zone 1 of all experimental units as described above. The fluid volume between wells in the experimental unit was balanced to prevent mass transfer of tau material between wells. The pathological tau was rapidly incorporated into the culture, and after 9 days of in vitro culture, a modulating antibody was added to zone 2 of all experimental units to regulate the proliferation of tau lesions in the culture. Again, the fluid volume was balanced to ensure that the antibody material did not transfer between wells in the experimental unit. During 14-16 day in vitro culture, synaptic function was evaluated in Zone 3 of all experimental units within the plate by analyzing calcium fluorescence transients. Subsequently, the cultures were fixed, stained for β-tubulin type 3 and endogenous tau (MAPT), and high-resolution images were acquired using a high-content imager. The impact of the cultures on synaptic function, along with the impact on network integrity and endogenous tau levels analyzed by automated image analysis, enabled high-volume screening of regulators of tauopathy progression.

[0047] To the best of our knowledge, this approach exhibits sufficient capacity and robustness to enable screening and profiling of a larger set of compounds in the search for molecules that prevent the diffusion of NDAPs across synaptic neurons.

[0048] Example 2 - Assay for monitoring retrograde axonal transport in primary cortical neurons The appearance and diffusion of misfolded tau and α-synuclein (α-syn) proteins within patient brain cells is a common pathological feature in neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. In vitro models provide evidence of interneuronal migration of α-syn, where the migrated protein acts as a seed to induce aggregation of endogenous proteins in recipient neurons. This diffusion of α-syn depends on several processes, including cellular uptake, axonal transport, somatic cell accumulation of seeds, and aggregation of endogenous proteins. Here, we evaluated retrograde axonal transport and cellular consequences of α-syn in primary neuronal cells using a novel microfluidic substrate.

[0049] Assay concept The assay is based on a 384-well microtiter plate containing 96 experimental units. Each unit consists of three spatially separated wells connected by microfluidic channels. The channels are small enough to prevent cell migration between wells. The inventors use co-culture plates to create compartmentalized cell cultures and local interventions to monitor and manipulate tau and α-syn axonal transport, somatic cell accumulation, and neuronal pathologies (e.g., protein aggregation).

[0050] method To achieve compartmentalized neuron culture, primary cortical cells derived from mouse embryonic (E18) brain tissue were cultured in only one well of the experimental unit, while the interconnected wells were left cell-free. After 6 days of in vitro culture (DIV), either fluorescently labeled, sonicated human α-synfibrillary cells (4 μg / ml, StressMarq Biosciences) were added to the cell-free wells. To establish a hydrodynamic barrier, the excess volume of well B relative to well A was 50 μl. Seven days after seed application, plates were fixed, antibody-stained, imaged (PerkinElmer, Operetta), and quantitatively evaluated.

[0051] result Characteristics of compartmentalized neuron cultures: Confocal images (20x magnification) show that axons that are βTub3+ but MAP2AB- grow through microchannels to interconnected well B, but the cell bodies and dendrites of MAP2AB+ neurons do not traverse to well B. (Figure 9A) Immunocytochemical staining allowed the inventors to characterize the cellular composition of the cortical cultures, demonstrating that the microfluidic substrate can maintain the same culture as a standard microtiter plate. The cellular composition of the cortical cultures was as follows: approximately 20,000 viable cells per well, 90% NeuN+ neurons, and 75% ± 6% PAX-6+ excitatory neurons.

[0052] α-syn seeds are taken up by axons and transported retrogradely to cells in well A, and high-content imaging shows the localization of fluorescently labeled α-syn-633 7 days after seed application. The 20x image in Figure 9B shows the uptake of fluorescently labeled α-syn-633 seeds by the axonal network in well B and the localization of fluorescently labeled α-syn-633 seeds in the cortical cell population in well A. A detailed confocal image (40x) shows that the seeds are taken up by axons in well B and transported retrogradely towards the cell bodies in well A.

[0053] Protein aggregation of endogenous proteins occurred in α-syn seed-filled neurons, and seven days after application of α-syn seeds, confocal imaging demonstrated that NeuN+ neurons filled with fluorescently labeled α-syn seeds showed phosphorylated endogenous α-synuclein (anti-α-syn, Abcam, clone [EP1536Y]) visible as neurite speckles and perinuclear accumulation (Figure 9C). Detailed confocal imaging showed co-localization of fluorescently labeled α-syn seeds and phosphorylated endogenous α-synuclein within NeuN+ neurons. Conclusion: We have established neuronal compartmentalization on our high-volume platform, enabling monitoring of cellular processes involved in α-synucleinopathy. Using microfluidic multiwell plates, we demonstrate that α-syn seeds are taken up by axons, transported retrogradely, and accumulate in the cell bodies of neurons, where they induce aggregation of endogenous proteins.

[0054] Example 3 - Predictive Example - Assay for Real-Time Monitoring of Axonal Transport Processes in Primary Cortical Neurons There is evidence that defects in various transport processes in neurons, such as mitochondrial transport defects, are contributing factors in neurodegenerative diseases such as Parkinson's disease (Sterky et al, Proc. Natl. Acad. Sci. USA 108, 12937-12942 (2011)). This is further supported by the fact that this phenomenon can be reproduced and regulated in in vitro disease models. However, MTS and / or HTS of mitochondrial transport processes along a single axon cannot be performed using conventional dissociation culture systems (Yu et al, Hum. Mol. Genet. 20, 3227-3240 (2011)). In this example, we evaluated axonal transport in real time using a novel microfluidic substrate along with an automated live imaging high-content method.

[0055] Assay concept The assay is based on a 384-well microtiter plate containing 192 experimental units. Each unit consists of two spatially separated wells, wells 1 and 2, connected by a microfluidic channel (Figure 4 / 5). The channel in well 2 is primarily surrounded by a small region accessible via the microchannel, which primarily functions as the detection region. Detection is used to track organelles such as mitochondria or inserted tracer particles, with the aim of evaluating the regulation of transport dynamics in axons. It should be noted that the channel is small enough to prevent cell movement between wells but allows axonal penetration, enabling detection transport processes only in aligned axons. method Primary cortical or midbrain cells derived from mouse embryonic (E16-18) brain tissue can be cultured in only one well of the experimental unit, with interconnected wells remaining cell-free. Once the axons have sufficiently penetrated the channels and the culture has reached an appropriate level of maturity, transport dynamics in the axons of living cells can be tracked using automated time-lapse confocal microscopy to track labeled endogenous structures or inserted particles. Depending on the cell model and the desired level of maturity of the culture, this assay can be performed at any point between 2 and 7 weeks after culturing. In one example, transport dynamics in axons are tracked by labeling mitochondria with mitochondrial dyes or by labeling fluorescent proteins with lentiviral particles and tracking these transport dynamics over a defined period. Typically, transport dynamics are tracked at 5-second intervals over a period of 3-5 minutes, after which, for example, transport rate, the ratio of retrograde to anterograde transport of mitochondrial particles in the assay, etc., can be evaluated. This method can be used to evaluate the regulation of transport dynamics in disease models and wild-type cultures in MTS and HTS applications. References JPEG0007893748000001.jpg229163

Claims

1. A multiwell plate comprising at least 96 directly accessible open wells having an optically transparent bottom, The multiwell plate comprises a substrate made from a material selected from the group consisting of polystyrene (PS), cycloolefin copolymer (COC) or cycloolefin polymer (COP), poly(methyl methacrylate) (PMMA), polycarbonate (PC), polyethylene (PE), polyethylene terephthalate (PET), polyamide, polypropylene, polyether ether ketone (PEEK), Teflon®, polydimethylsiloxane (PDMS), thermosetting polyester (TPE), silicon, glass, ceramic materials, and alumina. The multiwell plate comprises multiple layers, The surface properties of the multiwell plate enable the culture of adherent neuron cells, and the cells are directly accessible for chemical and physical manipulation. At least two wells in the plate are directly fluid-connected via at least one closed microchannel, the at least one closed microchannel is located at the boundary of the layer in the plane of the bottom of the well, the closed microchannel is defined by a groove in the layer of the plate and surrounded by the bottom layer of the plate, having a cross-sectional dimension (H and / or W) of 1 to 20 μm, preventing cell migration by flow or molecular diffusion between wells and minimizing mass migration, the fluid connection is formed between at least two non-adjacent wells, the fluid connection traverses the bottom of a well adjacent to at least one of the fluid-connected wells, and the plate further includes an optical detection marker to facilitate microscope autofocus to at least one well that is fluid-connected to another well. The surface of the substrate is optionally coated or chemically modified with a material selected from the group consisting of poly-l-lysine, poly-L-ornithine, collagen, laminin, Matrigel®, bovine serum albumin, poly[carboxybetaine methacrylate] (PCBMA), poly[[2-methacryloyloxy]ethyl]trimethylammonium chloride] (PMETAC), poly[poly(ethylene glycol)methyl ether methacrylate] (PPEGMA), poly[2-hydroxyethyl methacrylate] (PHEMA), poly[3-sulfopropyl methacrylate] (PSPMA), and poly[2-(methacryloyloxy)ethyldimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH). Multiwell plate.

2. A multiwell plate according to claim 1, wherein a fluid connection is formed between at least two adjacent wells.

3. The multiwell plate according to claim 1, wherein the multiwell plate conforms to the ANSI / SLAS Microplate Standard of the American National Standards Institute.

4. A multiwell plate according to any one of claims 1 to 3, wherein the material of the substrate includes polystyrene (PS), cycloolefin copolymer (COC), cycloolefin polymer (COP), poly(methyl methacrylate) (PMMA), polycarbonate (PC), polyethylene (PE), polyethylene terephthalate (PET), polyamide (Nylon®), polypropylene, polyetheretherketone (PEEK), Teflon®, PDMS, and / or thermosetting polyester (TPE).

5. The multiwell plate according to any one of claims 1 to 4, wherein the multiwell plate comprises a substrate made from cycloolefin copolymer (COP), cycloolefin polymer (COC), or polystyrene (PS).

6. The multiwell plate according to any one of claims 1 to 5, wherein the multiwell plate includes a substrate made of silicon, glass, ceramic material, or alumina.

7. The multiwell plate according to any one of claims 1 to 6, wherein the plate comprises a substrate having two or more layers, and optionally the layers are joined by ultrasonic welding, thermocompression bonding, plasma bonding, solvent-assisted bonding, laser-assisted bonding, or adhesive bonding using an adhesive or double-layer adhesive tape.

8. The multiwell plate according to any one of claims 1 to 7, wherein the plate comprises a substrate coated with a protein or polymer.

9. The multiwell plate according to claim 8, wherein the plate comprises a substrate coated with one or more of the following: poly-l-lysine, poly-L-ornithine, collagen, laminin, Matrigel®, or bovine serum albumin.

10. The multiwell plate according to any one of claims 1 to 9, wherein the plate comprises a substrate having a surface chemically modified with one or more of the following: poly[carboxybetaine methacrylate] (PCBMA), poly[[2-methacryloyloxy]ethyl]trimethylammonium chloride] (PMETAC), poly[poly(ethylene glycol)methyl ether methacrylate] (PPEGMA), poly[2-hydroxyethyl methacrylate] (PHEMA), poly[3-sulfopropyl methacrylate] (PSPMA), and poly[2-(methacryloyloxy)ethyldimethyl-(3-sulfopropyl)ammonium hydroxide] (PMEDSAH).

11. The multiwell plate according to any one of claims 1 to 10, wherein the plate comprises groups of at least two, at least four, at least eight, at least sixteen, at least 32, or at least 96 fluid-connected wells.

12. The multiwell plate according to any one of claims 1 to 11, wherein the at least one fluid connection portion includes a cross-sectional dimension (H and / or W) of 1 to 20 μm, such as 1 to 5 μm, 1 to 10 μm, 5 to 10 μm, 10 to 20 μm, 10 to 15 μm, 15 to 20 μm, or 5 to 15 μm, or includes a cross-sectional dimension (H and / or W) of 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, or 20 μm, and optionally also has an aspect ratio (H × W) in the range of 1:5 to 2:

1.

13. The multiwell plate according to any one of claims 1 to 12, wherein the fluid connection portion includes a cross-sectional dimension of 5 × 5 μm or less, or 3 × 3 μm to 5 × 5 μm.

14. The multiwell plate according to claim 13, wherein the dimensions, shape, and number of the fluid connections vary over the length of at least one fluid connection to improve nerve process penetration and productivity.

15. The multiwell plate according to any one of claims 1 to 14, wherein the length of the at least one fluid connection portion is at least 0.25 mm and at most 5.0 mm.

16. The multiwell plate according to any one of claims 1 to 15, wherein the aspect ratio of the dimensions of at least one fluid connection portion is in the range of 20:1 (W:H) to 1:5 (W:H).

17. The multiwell plate according to any one of claims 1 to 16, wherein the multiwell plate comprises a 96, 384, 1536, or 3456-well format and is optionally organized into a 2:3 rectangular matrix.

18. The multiwell plate according to any one of claims 1 to 17, wherein the multiwell plate comprises at least two groups of three adjacent, fluidly interconnected wells.

19. The multiwell plate according to any one of claims 1 to 18, wherein the multiwell plate includes at least three groups of two adjacent fluidically interconnected wells.

20. The multiwell plate according to any one of claims 1 to 19, wherein the multiwell plate includes at least one group of four adjacent, fluidly interconnected wells.

21. A screening method for high-throughput screening of a material of interest, comprising screening the material of interest using a multiwell plate according to any one of claims 1 to 20.

22. The method according to claim 21, wherein the material for the purpose is a 2D cell culture.

23. The method according to claim 21, wherein the material for the purpose is a 3D cell culture.

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