WO2024083938A1

WO2024083938A1 - System for cell culture and analysis comprising microfluidic plate

Info

Publication number: WO2024083938A1
Application number: PCT/EP2023/079047
Authority: WO
Inventors: Claudio Franco; Catarina FONSECA; Vania SILVERIO; Susana CARDOSO DE FREITAS; David BARATA
Original assignee: Instituto de Medicina Molecular João Lobo Antunes; Instituto De Engenharia De Sistemas E Computadores Para Os Microsistemas E As Nanotecnologias (Inesc-Mn); Instituto Superior Técnico
Priority date: 2022-10-20
Filing date: 2023-10-18
Publication date: 2024-04-25

Abstract

A system for cell culture and analysis and a method of analyzing the effect of shear stress on cells, the system comprising: a fluidic plate comprising at least one flow channel, each flow channel comprising an inlet, an enclosed passage and an outlet, wherein the enclosed passage is tapered at both ends, and at least one insert hole which forms an opening between the enclosed passage and the bottom of the fluidic plate; a culture plate comprising at least one through-hole; and an island plate comprising at least one raised island. The through-hole, the raised island and the insert hole are dimensioned such that the raised island can be inserted into the through-hole or the insert hole to form a liquid proof seal.

Description

SYSTEM FOR CELL CULTURE AND ANALYSIS COMPRISING MICROFLUIDIC PLATE

Field

The present invention relates to microfluidic systems for cell culture and analysis.

Background

Endothelial cells (ECs), the cells lining the interior of blood vessels, are constantly subjected to mechanical forces exerted by blood flow. Wall shear stress (WSS), the frictional force parallel to the vessel wall, exerts an influence on EC biology. ECs are able to sense WSS and convert it into a chemical response, by mechanotransduction (Dorland & Huveneers, Cellular and Molecular Life Sciences 74: 279-292, 2017). In response to blood flow, ECs reorganise and reorientate their cytoskeleton, elongating and aligning parallel to the flow direction. The response to blood flow also includes alteration of protein localisation at the cell membrane and modification of their gene expression profile. Two striking examples are the upregulation of the Krupple-like factors (KLF)2 and KLF4 transcription factors (Parmar et al., Journal of Clinical Investigation 116(1): 49-58, 2006; Atkins & Jain, Circulation Research 100: 1686-1695, 2007), and the polarisation and migration of ECs against the flow direction, a phenomenon known as flow-migration coupling.

ECs’ response to WSS is fundamental for vascular morphogenesis and its physiology. The hierarchical architecture of blood vessels depends on blood flow. ECs discriminate levels of shear stress, migrating from low-flow segments towards high-flow segments, leading to regression of poorly perfused vessels. Angiogenic ECs (tip cells) migrate against the flow direction toward emerging arteries, contributing to their growth. Loss of ALK1-signaling in ECs impairs flow-migration coupling leading to EC accumulation in capillaries, which results in the formation of arteriovenous malformations (Park et al 2021). Furthermore, the presence of disturbed and unstable flow in vessel bifurcations is associated with a pro-inflammatory stimulus promoting atherosclerosis, whilst laminar flow is a strong pro-quiescence and anti-inflammatory stimulus, an effect linked to the expression levels of KLF2/4, NO production and active NF-KB levels (Parmar et al., supra), all regulated by blood flow.

Although several cell surface molecules and structures have been shown to be mechanosensitive to WSS, e.g. ion channels and primary cilia, a clear understanding of the molecular mechanisms that regulate the diversity of EC flow responses remains lacking. To elucidate these mechanisms, there is a need for fluidic systems allowing genome-wide high- throughput screenings of ECs under flow. A variety of platforms to study the effects of shear stress in EC have been developed, such as cone-and-plate devices, orbital shakers, parallel flow plates and microfluidic channels.

In cone-and-plate devices, shear stress is applied to EC monolayers by the rotation of a cone or a disk that induces media flow (see e.g. Spruell & Baker, Biotechnology and Bioengineering 110(6): 1782-1793, 2013). The levels of shear stress applied in this system can be altered by increasing or decreasing the velocity of the cone. Although the use of cone-and-plate devices allows the performance of high-throughput siRNA screenings, such devices can create different shear stress levels across the monolayer of EC, ranging from zero in the centre to a maximum at the periphery, which can make this type of device unsuitable to study biological processes associated with response to flow (Hosseini et al., Lab on a Chip 21: 641-659, 2021).

Orbital shakers are also commonly used to combine various cell culture and biological assay applications and have been used to study the chronic effects of shear stress (Warboys et al., Atherosclerosis 285: 170-177, 2019). However, inertia effects within agitated wells result in complex flow profiles which make it difficult to characterise the shear stress experienced by the cells. Moreover, the cells experience different flow stress across the culture plate, and estimation of the local conditions for each well is challenging (Hosseini et al., supra). There are therefore disadvantages associated with using orbital shakers in this context.

Microfluidic cell cultures channels have become increasingly popular in the last decade (Rothbauer et al., Lab on a Chip 18: 249-270, 2018). Cells can be cultured inside a channel that mimics a segment of a blood vessel wall and perfusion methods can be applied to mimic blood flow. Several variations of parallel flow plates have been developed and although it is possible to apply constant laminar flow rates at physiological levels, these systems only permit the study of a very small number of genes, proteins, or conditions, lacking high-throughput capacity/capability. These characteristics make these systems incompatible with a screening approach, which would be very expensive and timeconsuming. Efforts have been made to improve the miniaturization of siRNA, DNA transfection or drug screenings using microfluidic lab-on-chips. However, the majority of these cellular microarray- and microchip-based methods use an open cell culture system, where cell media and secreted cellular factors are shared between different experimental conditions. Additionally, some of these devices lack the throughput capacity in terms of the number of parallel experimental conditions that can be set up or lack continuous laminar flow through the channels.

For instance, Schudel et al. developed a microfluidic system to perform siRNA screening to study cellular pathways involved in virus infection (Lab on a Chip 13: 811-817, 2013). Although the device has separate wells each with an inlet and outlet, the small volume of medium per channel and small channel size (400 pm) results in a small region with laminar shear stress allowing the analysis of only a small number of cells. Additionally, the small volume of medium used in this device may not be sufficient to generate physiological levels of shear stress.

Sinha et al. developed a device that combines both surface strain and shear stress on the cells with a flow array component containing multiple channels. In this device, cells are seeded on top of pillars, and the shape of the channels allows different levels of shear stress. However, since the flow channels are assembled before cell culture, it is impossible to perform siRNA transfection in a high-throughput manner using this setup (Sinha et al., Lab on a Chip 15: 429-439, 2015; Sinha et al., Scientific Reports 6: 29510, 2016).

A 384-well system was design by Xu and colleagues, to perform high-throughput RNAi screening to investigate cell mechanosensation. Here, 384 flat-headed pistons driven by an acoustic transducer were used to create shear stress in each well of a plate by moving the piston up and down with a given amplitude (Xu et al., Cell 173: 762-775, 2018). Although this provides a high-throughput system to perform siRNA screening, the movement of the piston generates disturbed fluid motion with an oscillatory pattern and non-uniform laminar flow.

More recently, Wei and colleagues reported a new system to perfuse regular 96-well plates. The advantage of this system (as with the system developed by Xu et al.), is the possibility of using regular cell culture plates for cell culture and performing siRNA transfection in a high-throughput and automated manner (Wei et al., Lab on a Chip 20: 4031- 4042, 2020). In addition, the authors showed that it is possible to connect the flow channels to a perfusion pump and recirculate the fluid through the system. For this purpose, the authors designed a fluidic component made of polydimethylsiloxane (PDMS) with several channels that feed each well of the plate. However, the medium that flows through the channels enters and exits the plate through a single inlet and a single outlet. Thus, this system has similar limitations to the system designed by Sinha and colleagues (supra), in that it is not possible to test different flow conditions in the different channels. In addition, the medium that flows through the wells is turbulent in its profile and creates a gradient of shear stress generating a diversity of flow responses, and only very low shear stress can be achieved.

Thus, there is a need for a system that combines an ability to study a very large number of genes, proteins, or conditions with an ability to expose cells (which have been previously manipulated, e.g. by RNAi) to a constant and laminar flow profile, at a physiological level. Provided herein is such a system. Summary of Invention

In a first aspect, provided herein is a system for cell culture and analysis comprising a fluidic plate, the fluidic plate comprising at least one flow channel, each flow channel comprising an inlet, an enclosed passage and an outlet; wherein the inlet and outlet are located at either end of the enclosed passage, and each forms an opening between the enclosed passage and a side of the fluidic plate; wherein the enclosed passage of the at least one flow channel is tapered at both ends, and the inlet and outlet open into the tapered ends of the enclosed passage.

In particular embodiments of the first aspect, the fluidic plate of the system for cell culture and analysis of the first aspect further comprises at least one insert hole which forms an opening between the enclosed passage and the bottom of the fluidic plate, wherein the at least one insert hole is located between the inlet and the outlet; and the system further comprises:

(i) a culture plate, said culture plate comprising at least one through-hole; and

(ii) an island plate, comprising at least one raised island; wherein the through-hole, the raised island and the insert hole are dimensioned such that the raised island can be inserted into the through-hole or the insert hole to form a liquidproof seal; and such that insertion of the raised island into the through-hole forms a well suitable for cell culture, and such that upon insertion of the raised island into the insert hole the top of the raised island is level with the base of the enclosed passage such that the base of the enclosed passage is an essentially flat surface; and wherein the culture plate, island plate and fluidic plate are configured such that the culture plate can be placed on top of the island plate such that the one or more raised islands insert into the one or more through-holes, thereby forming a cell culture configuration; and such that the fluidic plate can be placed on top of the island plate such that the one or more raised islands insert into the one or more insert holes, thereby forming a fluidic configuration.

In a second aspect, provided herein is a system for cell culture and analysis, the system comprising:

(i) a culture plate, said culture plate comprising at least one through-hole;

(ii) an island plate, comprising at least one raised island; and

(iii) a fluidic plate, comprising at least one flow channel, each flow channel comprising an inlet, an enclosed passage and an outlet, and further comprising at least one insert hole; wherein the inlet and outlet are located at either end of the enclosed passage, and each forms an opening between the enclosed passage and a side of the fluidic plate; and the insert hole forms an opening between the enclosed passage and the bottom of the fluidic plate, and the at least one insert hole is located between the inlet and the outlet; wherein the through-hole, the raised island and the insert hole are dimensioned such that the raised island can be inserted into the through-hole or the insert hole to form a liquidproof seal; and such that insertion of the raised island into the through-hole forms a well suitable for cell culture, and such that upon insertion of the raised island into the insert hole the top of the raised island is level with the base of the enclosed passage such that the base of the enclosed passage is an essentially flat surface; and wherein the culture plate, island plate and fluidic plate are configured such that the culture plate can be placed on top of the island plate, such that the one or more raised islands insert into the one or more through-holes, thereby forming a cell culture configuration; and such that the fluidic plate can be placed on top of the island plate such that the one or more raised islands insert into the one or more insert holes, thereby forming a fluidic configuration.

In a third aspect, provided herein is a kit comprising a culture plate, an island plate and a fluidic plate as defined in the first or second aspect.

In a fourth aspect, provided herein is a method of analysing the effect of shear stress on cells, comprising:

(i) providing, and optionally sterilising, a culture plate, an island plate and a fluidic plate, each as defined in the first or second aspect;

(ii) assembling a cell culture configuration by placing the culture plate upon the island plate;

(iii) seeding the cells into the wells and culturing the cells in medium in said wells;

(iv) removing the medium from the wells;

(v) disassembling the cell culture configuration by removing the culture plate from the island plate;

(vi) assembling the fluidic configuration by placing the fluidic plate upon the island plate;

(vii) applying a continuous flow of fluid through the one or more flow channels, wherein the fluid enters each flow channel through the inlet, passes through the enclosed passage and exits the flow channel through the outlet; and

(viii) analysing the cells adhered to the raised islands to determine the effect of shear stress on the cells.

Detailed Description

The present invention provides a system for cell culture and analysis. Specifically, the system is for analysis of the response of cells (particularly endothelial cells) to shear stress resulting from the flow of fluid across the cells. The system can thus be used as a model for blood flow through blood vessels. The system comprises at least a fluidic plate which, as described further below, comprises at least one flow-channel. The fluidic plate may be used alone, in which case cells for analysis may be adhered to the interior of the flow-channel, and optionally cultured, before a liquid is passed through the flow channel such that it flows across the adhered cells.

Alternatively, and as described further below, the system may comprise three primary components: a culture plate, an island plate and a fluidic plate. To use the system in this context, a cell culture configuration is first assembled from the culture plate and island plate. The cell culture configuration comprises wells for cell culture, the wells being formed by through-holes present in the culture plate, with the bases of the wells being formed by the tops of raised islands present on the island plate. Cells are seeded into the wells of the cell culture configuration and are cultured so that they adhere to the bases of the wells, i.e. to the tops of the raised islands. During cell culture, the cells may be treated with an agent of interest to test the impact of that agent on the cells’ response to shear stress. For instance, the cells may be transfected with an RNAi agent, such as a small interfering RNA (siRNA), to knock down expression of a gene of interest, to investigate the role of that gene in the cellular shear stress response. Subsequently, the culture medium is removed from the wells and the cell culture configuration disassembled. A fluidic configuration is then assembled from the fluidic plate and island plate. The fluidic configuration comprises multiple flow channels, the bases of which are partially formed by the tops of the raised islands of the island plate. Fluid is passed through the flow channels, and the fluidic configuration then disassembled.

Phenotypic characterisation of the cells can then be performed using a wide range of molecular biology techniques. For example: the cells can be detached or lysed on a perisland basis to analyse their DNA, RNA or protein contents; or the cells can be fixed using different fixation reagents and subsequently used for in situ hybridisation, immunohistochemistry or immunofluorescence to analyse protein or RNA localization. In addition, the fluidic plate can be designed to allow imaging through the top, allowing for live- imaging modalities of fluorescent reporters of biological activities. Thus, the invention allows the impact of shear stress on cells to be determined at multiple levels.

A particular advantage of the system of the invention is that the design of the fluidic plate ensures a steady, laminar flow of fluid through the plate. This ensures that the cells on top of all the islands are exposed to uniform, high levels of shear stress. A difficulty associated with many existing devices for shear stress analysis is that, as described in the Introduction, flow across or around the cells of interest is not steady and laminar, meaning that the cells of interest are not exposed to uniform levels of shear stress, which is problematic for the analysis of experimental results obtained from these devices. Thus the system of the invention provides a high-throughput means for analysis of the cellular response to shear stress.

As set out above, the culture plate, island plate and fluidic plate of the system respectively comprise at least one through-hole, at least one raised island, and at least one flow channel comprising at least one insert hole. Accordingly, all references to through- holes, raised islands, flow channels and insert holes, whether singular or plural, are to be understood as encompassing both the singular and the plural (dependent on the design of the plates), unless the context clearly dictates otherwise.

The culture plate comprises at least one through-hole. By through-hole is meant a hole running from one side of the plate to the other (or passing from one side of the plate to the other). That is to say, an opening is present on two sides of the plate, between which the through-hole runs. The through-hole runs from one side of the plate to the opposite side of the plate. In the case of the culture plate, the sides of the plate comprising the through-hole openings are defined as the top and bottom of the culture plate. The plate may be symmetrical such that the top and bottom of the plate are identical and interchangeable. Alternatively, these two sides of the culture plate may have distinguishing features, as set out below.

Preferably, the top and bottom of the culture plate are both essentially flat, i.e. nonsloping (though raised or indented features may be present) and are parallel to each other, and the through-hole is perpendicular (or essentially perpendicular) to the top and bottom of the culture plate, i.e. the through-hole runs straight through the culture plate from top to bottom, without bends or turns.

Preferably, the culture plate comprises multiple through-holes, as further discussed below. In this case, all of the through-holes are preferably of the same size and shape (though this is not essential, so long as they are matched to the raised islands on the island plate, as detailed further below). When multiple through-holes are present, all the through- holes run between the top and bottom of the plate (i.e. the multiple through-holes are parallel to each other), and the through-holes are preferably arranged in a regular fashion, e.g. in rows and columns. However, any arrangement of through-holes can be used so long as it is matched to the arrangement of raised islands on the island plate.

The through-holes of the culture plate may be of any shape and size. However, as set out above, these must form wells when the culture plate and island plate are assembled into the cell culture configuration.

The island plate comprises at least one raised island. The raised island is a protrusion which projects from one side of the island plate. Preferably, the island plate comprises multiple raised islands, in which case all the raised islands project from the same side of the island plate. The side of the island plate from which the raised islands project is defined as the top of the island plate. Preferably, the top of the island plate is essentially flat (i.e. non-sloping), with protruding raised islands, essentially perpendicular to the top of the island plate. The bottom of the island plate (i.e. the side opposite to the top of the island plate) may also be essentially flat, and parallel to the top. The island plate can thus be seen as comprising a base section from which the raised islands protrude.

The tops of the raised islands are flat, i.e. not sloped, convex or concave. Preferably, the raised islands are all of the same shape and width/diameter. The raised islands are preferably all of the same height. As detailed above, the island plate is assembled with the culture plate to form the cell culture configuration, and with the fluidic plate to form the fluidic configuration. Thus the shape and size of the raised islands must be matched to those of the through-holes of the culture plate and the insert holes of the fluidic plate. Where multiple raised islands are present on the island plate, preferably they are arranged in a regular fashion, e.g. in rows and columns, but any arrangement of raised islands can be used, so long as it matches the arrangement of the through-holes of the culture plate and the insert holes of the fluidic plate.

The fluidic plate comprises at least one flow-channel. A flow channel is a channel through which a fluid (i.e. liquid) can pass. Preferably, a flow channel is straight (i.e. without curves or bends) and has an essentially flat internal surface (i.e. the internal surface is preferably non-sloping), lacking raised parts or indentations. The flow channel comprises two holes, one located at each end of the channel, which run from one side of the fluidic plate to the flow channel and constitute an inlet and an outlet, through which liquid can pass and, respectively, enter or exit the flow channel. The inlet and outlet may be of any suitable shape and size, as discussed further below. The inlet and outlet may be identical and thus interchangeable, or may have distinguishing features to render the inlet and outlet distinct from each other.

Running between the inlet and outlet is an enclosed passage. Together with the inlet and outlet the enclosed passage forms the flow channel. The inlet and outlet each form an opening between the enclosed passage and a side of the fluidic plate. Fluid can pass through the flow channel by entering through the inlet, passing through the enclosed passage and exiting through the outlet. The inlet and outlet may both form an opening on one side of the fluidic plate, or alternatively the inlet may form an opening on one side of the fluidic plate and the outlet form an opening on the other side of the fluidic plate. That the enclosed passage is ‘enclosed’ means that during operation of the fluidic plate (i.e. when liquid is flowing through it), the inlet and outlet are the only openings into or out of the flow channel through which fluid can pass.

The fluidic plate preferably comprises multiple flow channels. The multiple flow channels are preferably arranged essentially parallel to each other, and are preferably of the same length, with the inlets and outlets arranged in a row or column. Preferably, all inlets open onto the same side of the fluidic plate and all outlets open onto the same side of the fluidic plate. The fact that each flow channel has its own inlet and outlet (rather than the plate comprising a single inlet and single outlet shared by all flow channels) is advantageous as it means different conditions can be applied to each individual channel during experimentation.

Each flow channel comprises at least one hole located in the central region of the fluidic channel, between the inlet and outlet, referred to herein as an insert hole. The insert hole runs from the enclosed passage of the fluidic channel to the side of the fluidic plate, thus forming an opening on the side of the fluidic plate into the central region of the enclosed passage. The side of the fluidic plate onto which the insert hole opens is defined as the bottom of the fluidic plate, and the opposite side is the top of the fluidic plate. Preferably, the top and bottom of the fluidic plate are both flat (i.e. non-sloping) and are parallel to each other.

As set out above, the insert hole is designed such that a raised island can be inserted into it. The insert hole may thus be of any shape or size, so long as it matches with the raised island. The insert hole is essentially perpendicular to the bottom of the fluidic plate.

Preferably, each flow channel comprises multiple insert holes, which are preferably evenly spaced in a row along the channel. Preferably, each flow channel comprises the same number of insert holes, which are preferably identically arranged in each flow channel.

As noted above, in some embodiments the inlet and outlet of the flow channel open onto any side of the fluidic plate. The inlet and outlet may open onto the top of the fluidic plate. In this case the inlet and outlet may be perpendicular to the top of the fluidic plate, or may be sloped into the enclosed passage. Alternatively, the inlet and outlet open onto the bottom of the fluidic plate. Again, in this case the inlet and outlet may be perpendicular to the bottom of the fluidic plate, or may be sloped into the enclosed passage. In another alternative, the inlets and outlets may open onto lateral sides of the fluidic plate (i.e. onto sides which are neither the top nor the bottom of the plate). In this case, the inlet and outlet may be arranged at any suitable angle both horizontally and vertically relative to the enclosed passage.

In other embodiments, the inlet and outlet open onto different sides of the fluidic plate, e.g. one may open onto the top and the other the bottom, or one may open onto the top and the other onto a lateral side. If the inlet and outlet open onto a lateral side of the fluidic plate, they may open onto the same lateral side or different lateral sides. Preferably however, all inlets and outlets open onto the top of the fluidic plate.

As set out above, during use of the system the island plate is assembled first into a cell culture configuration, in combination with the culture plate, and then into a fluidic configuration, in combination with the fluidic plate. Many features of the island plate, culture plate and fluidic plate are therefore interdependent.

To form the cell culture configuration, the raised islands of the island plate are inserted into the through-holes of the culture plate to form wells for cell culture. To generate suitable wells it is necessary for a liquid-proof seal to be formed upon insertion of a raised island into a through-hole, to avoid leakage of culture medium during cell culture. Similarly, the raised islands of the island plate are inserted into the insert holes of the fluidic plate to expose adhered cells to flow. Insertion of the raised island into the insert hole must form a liquid-proof seal to avoid fluid leakage out of the flow channel. The through-hole, insert hole and raised island must therefore be dimensioned such that upon insertion of the raised island into the through-hole or insert hole a liquid-proof seal is formed.

To this end, the through-hole, insert hole and raised island are preferably all the same shape. By shape herein is meant the cross-sectional shape of the raised island on the axis essentially perpendicular to the top of the island plate, and the corresponding shape of the through-hole and insert hole. Any suitable shape may be selected for the through hole, insert hole and raised island, e.g. square, rectangle, circle, etc. The through-hole and insert hole have the same width (or diameter), appropriate to the width/diameter of the raised island. The raised island has essentially the same width/diameter as the through-hole and insert hole, though the raised island may be slightly narrower than the through-hole and insert hole, to allow its insertion into those holes.

The island plate, the culture plate and the fluidic plate may all be essentially the same size (that is to say they may have the same width and length, so that when the culture plate or fluidic plate is placed upon the island plate to form the cell culture configuration or fluidic configuration, the edges of the island plate essentially meet the edges of the culture plate or fluidic plate). In this case, the island plate preferably comprises the same number of raised islands as the culture plate comprises through-holes and the fluidic plate comprises insert holes, and the raised islands, through-holes and insert holes are arranged identically across their respective components, so that each raised island has a corresponding through- hole and insert hole, each through-hole has a corresponding raised island and each insert hole has a corresponding raised island.

It is not, however, essential for each of these three components to be of the same size, so long as their sizes and arrangements are such that it is possible to use at least one flow channel experimentally. In some embodiments, the island plate is larger than the culture and/or fluidic plates, and comprises multiple rows of raised islands. In this case, the culture and/or fluidic plates comprise a smaller number of rows of through-holes or insert holes (respectively). The smaller culture and/or fluidic plates can nonetheless be placed upon the island plate to form the cell culture or fluidic configuration. In a particular embodiment, multiple smaller culture and/or fluidic plates can be placed upon the island plate simultaneously. For instance, each culture plate or fluidic plate may comprise a single row of through-holes or insert holes, and thus a separate culture plate or fluidic plate can be placed on each row of raised islands on the island plate.

Alternatively, though less conveniently, the island plate may be smaller than the culture and/or fluidic plates, such that the culture and/or fluidic plates may be placed upon multiple island plates to assemble the cell culture and fluidic configurations.

In another, preferred alternative, discussed further below, the island plate is smaller than the culture and/or fluidic plate, due to the presence of a surround which extends outwards and downwards from the sides of the culture or/fluidic plate forming a cavity of corresponding dimensions to the island plate, such that when the culture and/or fluidic plate is placed upon the island plate the island plate is enclosed within the cavity (i.e. the culture/fluidic plate is on top of the island plate, and the surround covers the lateral sides of the island plate).

Regardless of the relative sizes of the various plates, in order that the culture plate and island plate can be assembled into the cell culture configuration, and the fluidic plate and island plate can be assembled into the fluidic configuration, the raised islands, through- holes and insert holes are arranged in their relative plates in matching arrangements which enable the necessary assemblies to be performed.

The height of the raised islands is also dependent on the height of the culture plate (and thus depth of the through-holes) and the depth of the insert holes (i.e. the distance from the bottom of the fluidic plate to the flow channel running through it).

As noted above, insertion of a raised island into a through-hole in the culture plate must form a well for cell culture. The depth of the well corresponds to the difference between the height of the raised island and the depth of the through-hole, e.g. if the through-hole is 1 cm deep and the raised island is 0.5 cm high the depth of the resulting well is 0.5 cm. The difference between the height of the raised island and the depth of the through-hole must be sufficient to yield a well deep enough for cell culture. Any depth of well may be used so long as it is suitable for cell culture. It is preferred that the well is at least 5 mm deep for the purpose of cell culture, e.g. at least 6 mm or at least 7 mm, though shallower wells may also be suitable. In some embodiments the well is 7 mm deep. In other embodiments the well is 7.5 mm deep. The well may be more than 7 mm deep, e.g. at least 8, 9 or 10 mm deep. In some embodiments, the well has a depth in the range of 10-12 mm, as for standard 96-well plates.

To form a well as specified above, the raised island and through-hole may be any suitable combination of height and depth. Preferably though, the height of the raised island and depth of the through-hole are minimised, while ensuring that the raised island is capable of providing a liquid-proof seal for the through-hole. Preferably, the raised island is at least

I mm high, e.g. at least 2, 3, 4 or 5 mm high. In some embodiments the raised island is 3 mm high.

The through-holes have a suitable depth to form a well with such a raised island. For instance, the through-holes may be at least 7, 8, 9, 10, 11 or 12 mm deep. In some embodiments, the through-hole is 10.5 mm deep. In other embodiments the through-hole is

I I mm deep. As is apparent from the fact that the through-hole passes all the way through the culture plate, the depth of the through-hole corresponds to the height (or thickness) of the culture plate. That is to say, the depth of the through-hole is the same as the height of the culture plate (from bottom to top).

In some embodiments, the raised island is 3 mm high and the through-hole is 11 mm deep, yielding cell culture wells of 8 mm. In other embodiments the raised island is 3 mm high and the through-hole is 10.5 mm deep, yielding cell culture wells of 7.5 mm.

As noted above, the island plate comprises a base from which the raised islands protrude. The base of the island plate may be of any height. For instance, it may be the same height as the raised islands. The base of the island plate may for instance be 2-5 mm high. In some embodiments the base of the island plate is 3 mm high (from bottom to top).

As further noted above, the raised islands and the insert holes are dimensioned such that when the raised islands are inserted into the insert holes of the fluidic plate, the top of the raised island is level with the base of the enclosed passage, such that the base of the enclosed passage is an essentially flat surface. The base of the enclosed passage is the side or section of the passage which forms the bottom of the enclosed passage during use, i.e. it is the side or section of the passage wall closest to the bottom of the fluidic plate. When inserted into the insert hole, the top of the raised island is level with the base of the enclosed passage, i.e. the top of the raised island is at the same height as the base of the enclosed passage. The base of the enclosed passage is thus essentially flat. In this instance this means that the base of the enclosed passage does not comprise raised or lowered sections (indentations). To achieve this, it is required that the depth of the insert hole is the same as the height of the raised island. For instance, if the raised island is 3 mm high, the insert hole must be 3 mm deep. The insert hole is thus preferably at least 2 mm deep, e.g. at least 3, 4 or 5 mm deep. In some embodiments the raised island is 3 mm high and the insert hole is 3 mm deep. As is apparent, the depth of the insert hole corresponds to the distance between the bottom of the fluidic plate and the base of the enclosed passage of the flow channel within the fluidic plate.

The island plate, culture plate and fluidic plate may be of any suitable shape and size. For instance, they may be square, rectangular or circular. Preferably the three plates are of the same or similar shape, though this is not essential so long as the raised islands can be aligned with both the through-holes of the culture plate and the insert holes of the fluidic plate. In some embodiments, the island plate, culture plate and fluidic plate are all essentially rectangular. By ‘essentially rectangular’ means that the shape of the plate is generally rectangular, but not necessarily perfectly rectangular. In some embodiments, as shown in the Examples, the island plate has a standard rectangular shape, while both the culture plate and the fluidic plate are essentially rectangular, having a generally rectangular shape with one ‘cut-off corner’, as is common in commercial tissue culture plates. Any one or more of the culture plate, the island plate and the fluidic plate may be essentially rectangular with one or more cut-off corners, preferably one cut-off corner.

The plates may be of any size and comprise any desired number of raised islands, through-holes and insert holes, respectively, so long as the plates are matching to each other and can be assembled into the cell culture and fluidic configurations. However, it is preferred that the plates are the size and design of a standard tissue culture plate, to enable their compatibility with other standard laboratory equipment. For instance, the plates may comprise, respectively, 12 raised islands, through-holes and insert holes, 24 raised islands, through-holes and insert holes, 48 raised islands, through-holes and insert holes, 96 raised islands, through-holes and insert holes, 384 raised islands, through-holes and insert holes, or 1536 raised islands, through holes and insert holes. The size of the raised islands, through-holes and insert holes is of course dependent on the number present (the more present, the smaller they must be).

The plates thus preferably have the dimensions of a standard tissue culture plate, as defined by the American National Standards Institute (ANSI) and the Society for Laboratory Automation and Screening (SLAS). The plates’ length is preferably in the range 125- 130 mm, preferably in the range 127-128 mm, most preferably in the range 127.7-127.8 mm, e.g. 127.74, 127.75 or 127.76 mm. Their width is preferably in the range 83-88 mm, preferably in the range 85-86 mm, most preferably in the range 85.4-85.5 mm, e.g. 85.4 or 85.48 mm. In some embodiments, the culture, island and fluidic plates all have the same dimensions or approximately the same dimensions.

In some embodiments, as discussed above, the fluidic plate and/or the culture plate (but particularly the fluidic plate), may comprise an internal section and a surround. The internal section comprises the through-holes or insert holes, and is encircled by the surround. The surround is thicker than the internal section and extends below the base of the internal section, forming a large cavity immediately below the internal section of the fluidic plate/culture plate. The cavity is the same depth as the island plate (excluding the raised islands) such that when the fluidic/culture plate is placed on the island plate, the raised islands insert into the through-holes or insert holes and the island plate fills the cavity. This advantageously improves the seal of the assembly of the island plate with the fluidic/culture plate and thus reduces leakage.

Thus the culture plate and/or fluidic plate may comprise a surround which extends below the internal section, forming a cavity dimensioned to house the island plate when the cell culture configuration or fluidic configuration is assembled. In some embodiments the fluidic plate comprises such a surround.

When the fluidic or culture plate comprises an internal section and surround, the internal section has the dimensions specified for tissue culture plates by the ANSI/SLAS, as set out above. The surround may have any width, e.g. 5-15 mm. The surround may have the same or different widths on each side of the plate. In some embodiments, the fluidic plate comprises a surround of about 13 mm with lengthways and about 8.5 mm widthways around an internal section of 127.74 x 85.4 mm, giving total dimensions of about 153 x 102 mm, as shown in the Examples. The thickness of the surround corresponds to the thickness of the internal section of the plate plus the thickness of the base section of the island plate.

In some preferred embodiments the culture plate comprises 96 through-holes, the island plate comprises 96 raised islands and the fluidic plate comprises 96 insert holes, the 96 through-holes, raised islands and insert holes arranged in the same manner as the wells of a 96-well plate according to ANSI/SLAS standards. In these embodiments, the fluidic plate comprises 8 or 12 flow channels, corresponding to the 8 rows or 12 columns of insert holes in the plate. Specifically, the fluidic plate comprises either (i) 8 flow channels each comprising 12 insert holes, or (ii) 12 channels each comprising 8 insert holes.

In other preferred embodiments the culture plate comprises 384 through-holes, the island plate comprises 384 raised islands and the fluidic plate comprises 384 insert holes, the 384 through-holes, raised islands and insert holes arranged in the same manner as the wells of a 384-well plate according to ANSI/SLAS standards. In these embodiments, the fluidic plate comprises 16 or 24 flow channels, corresponding to the 16 rows or 24 columns of insert holes in the plate. Specifically, the fluidic plate comprises either (i) 16 flow channels each comprising 24 insert holes, or (ii) 24 channels each comprising 16 insert holes.

However many through-holes, raised islands and insert holes are present in the plates, they preferably have the ANSI/SLAS standard dimensions of a well in a plate bearing the same number of wells. For instance, in the case that the plates have 96 through-holes, raised islands and insert holes, the through-holes, raised islands and insert holes may have diameters of 5-6 mm (if circular), or if square have 5x5-6x6 mm dimensions. In some embodiments the plates have 96 through-holes, raised islands and insert holes, each 6x6 mm square.

In the fluidic plate, the dimensions of the flow channel can be selected as desired/appropriate for the given experiment, and may accordingly be any value as considered suitable by the skilled person. For example, the height of the flow channel (i.e. of the enclosed passage) may be in the range from 0.1-1 mm, e.g. 0.1-0.9 mm, 0.2-0.8 mm, 0.3-0.7 mm or 0.4-0.6 mm. The flow channel may have a height of about 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 mm. Preferably the flow channel has a height of 0.5 mm or about 0.5 mm. For example, the width of the enclosed passage of the flow channel may be in the range 0.05-10 mm, e.g. 0.05-9 mm, 0.05-8 mm, 0.05- 7 mm, 0.05-6 mm, 0.05-5 mm, 0.05-4 mm, 0.05-3 mm, 0.05-2 mm, 0.05-1 mm, 0.01-10 mm, 0.1- 9 mm, 0.1-8 mm, 0.1-7 mm, 0.1-6 mm, 0.1-5 mm, 0.1-4 mm, 0.1-3 mm, 0.1-2 mm, 0.1-1 mm, 0.5-10 mm, 0.5-9 mm, 0.5- 8 mm, 0.5-7 mm, 0.5-6 mm, 0.5-5 mm, 0.5-4 mm, 0.5-3 mm, 0.5-2 mm, 0.5-1 mm, 1-10 mm, 1-9 mm, 1-8 mm, 1-7 mm, 1-6 mm, 1-5 mm, 1-4 mm, 1-3 mm, 1-2 mm, 2-10 mm, 2-9 mm, 2- 8 mm, 2-7 mm, 2-6 mm, 2-5 mm, 2-4 mm, 2-3 mm, 3-10 mm, 3-9 mm, 3-8 mm, 3-7 mm, 3-6 mm, 3-5 mm, 3-4 mm, 4-10 mm, 4-9 mm, 4-8 mm, 4-7 mm, 4-6 mm, 4-5 mm, 5-10 mm, 5-9 mm, 5-8 mm, 5-7 mm, 5-6 mm, 6-10 mm, 6-9 mm, 6-8 mm, 6-7 mm, 7-10 mm, 7-9 mm, 7-8 mm, 8-10 mm, 8-9 mm or 9-10 mm. For instance the enclosed passage of the flow channel may have a width of 1 , 2, 3, 4, 5 or 6 mm. In some embodiments the enclosed passage of the channel has a width of 6 mm.

The inlet and outlet are preferably circular and may have a diameter in the range 0.2-5 mm, e.g. 0.2-4 mm, 0.2-3 mm, 0.2-2 mm, 0.2-1 mm, 0.5-5 mm, 0.5-4 mm, 0.5-3 mm, 0.5-2 mm, 0.5-1 mm, 1-5 mm, 1-4 mm, 1-3 mm, 1-2 mm, 2-5 mm, 2-4 mm, 2-3 mm, 3-5 mm, 3-4 mm or 4-5 mm. For instance the inlet and/or outlet may have a diameter of about 0.5, 1 2, 3, 4 or 5 mm. The inlet and outlet may have the same diameter as each other, or may have different diameters. Preferably they have the same diameter. In some embodiments, the inlet and outlet both have a diameter of 4 mm or about 4 mm. The diameter of the inlet and outlet may be selected for compatibility with e.g. particular tubing or luer fitting or suchlike which is used to introduce the fluid into the flow channel.

The length of the flow channel depends on the length of the plate and the rotation of the flow channel in the plate (i.e. whether it is across the width or length of the plate). In some embodiments the flow channel is positioned across the length (i.e. the longer dimension) of the plate. Where the plate has a length (or an internal section with a length) of a standard tissue culture plate as set out above (i.e. about 127-128 mm), the flow channel may extend to within about 1 mm of each side of the plate or internal section thereof. Thus the flow channel may have a length in the range of 125-126 mm, e.g. 125.5-126 mm, e.g. 125.6-125.9 mm, e.g. 125.7-125.8 mm. In some embodiments the flow channel has a length of 125.77 mm.

In some preferred embodiments, the enclosed passage does not have a uniform width all the way along, but rather the width tapers (i.e. becomes narrower) at both ends. The inlet and outlet preferably open into the tapered ends of the enclosed passage. In preferred embodiments, the diameter of the inlet and outlet is greater than the width of the enclosed passage where the inlet and outlet meet the enclosed passage. It will be understood that this arrangement is particularly suitable when the inlet and outlet open onto the top or bottom of the fluidic plate. This arrangement of the diameters of the inlet and outlet and the width of the enclosed passage is advantageous as it has been found to reduce the entry of air into the system. Nonetheless, in other embodiments, the diameter of the inlet and outlet is less than the width of the enclosed passage where the inlet and outlet meet the closed passage, and in other embodiments the diameter of the inlet and outlet may be the same as the width of the enclosed passage where the inlet and outlet meet the enclosed passage.

The diameter of the inlet and outlet may thus be greater, less than or equal to the width of the enclosed passage where it meets the inlet and outlet, though it is preferred that the diameter of the inlet and outlet is greater than the width of the enclosed passage where they meet the enclosed passage. As noted above, the inlet and outlet are not required to be the same size and so while it is preferred that both have a diameter greater than the width of the enclosed passage where they meet the enclosed passage, it is possible that, e.g., one may have a diameter greater than and the other a diameter less than the width of the enclosed passage where they meet it.

Any difference in size between the diameter of the inlet or outlet and width of the enclosed passage where the inlet or outlet meets it may be selected as optimal for the particular experimental arrangement. For example, the diameter of the inlet and outlet may be in the range 50 % to 300 % of the width of the enclosed passage where the inlet or outlet meets the enclosed passage. For example, the diameter of the inlet and/or outlet may be SO- 75, 50-100, 50-125, 50-150, 50-175, 50-200, 50-250, 75-100, 75-125, 75-150, 75-175, 75- 200, 75-250, 75-300, 100-125, 100-150, 100-200, 100-250, 100-300, 125-150. 125-175, 125-200, 125-250, 125-300, 150-200, 150-250, 150-300 or 200-300 % of the width of the enclosed passage where it meets the inlet and outlet. For example, the diameter of the inlet and/or outlet may be about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275 or 300 % of the width of the enclosed passage where it meets the inlet and outlet.

Since the width of the enclosed passage may taper and in this instance the inlet and outlet preferably open into the tapered section of the enclosed passage, when the inlet and outlet have a diameter greater than the width of the enclosed passage where it meets the inlet and outlet, the diameter of the inlet and outlet may nonetheless by greater, less than or equal to the maximum width of the enclosed passage. For instance, the Example below shows an embodiment in which the flow channels have a maximum width of 6 mm, but a width of only 2 mm where they meet the inlet and outlet. The inlet and outlet both have a diameter of 4 mm, i.e. twice as big as (200 % of the width of) the enclosed passage where it meets the inlet and outlet, but smaller than the maximum width of the passage.

Where the enclosed passage has tapered ends, it has a continuous width along the majority of its length, including along the entirety of the length which comprises insert holes (or the tops of raised islands, when the fluidic plate is assembled into the fluidic configuration). For simplicity, this width may be referred to as the width at the midpoint of the enclosed channel. The midpoint of the enclosed passage is the point half-way along the length of the passage, i.e. the position equidistant from the two ends of the passage. As noted above, this width is not only the width of the midpoint, but is also the width of the enclosed passage along the majority of its length.

As noted, the enclosed passage is preferably tapered at both ends, and the inlet and outlet preferably open into the tapered sections of the enclosed passage. In such embodiments neither the inlet nor the outlet open onto the enclosed passage at a location where its width is continuous (i.e. where its sides are parallel to each other), but rather both the inlet and the outlet open onto the enclosed passage in the area where the width of the passage is tapering, i.e. narrowing. For simplicity, the ratio of the diameter of the inlet or outlet to the width of the enclosed passage is determined based on the width of the enclosed passage at the centre of the inlet or outlet.

The enclosed passage may taper to a point (which may be a curved point), in which case ultimately the two sides of the enclosed passage essentially meet.

Thus the enclosed passage is preferably tapered at both ends. When the enclosed passage has tapered ends, it may be that a central section of the enclosed passage has a continuous width. That is to say, the midpoint of the enclosed passage may also be the midpoint of the section of continuous width, and equal lengths of the enclosed passage may be tapered at each end. In this case, when the inlet and outlet have the same diameter, the midpoint of the enclosed passage is generally a plane of symmetry across the passage.

The enclosed passage may comprise a section of continuous length which constitutes in the range of 50-95 % of the total length of the enclosed passageway, and thus correspondingly tapered sections which constitute in the range of 5-50 % of the total length of the enclosed passageway. As noted above, the section of continuous length is preferably a central section, such that the tapered sections at each end are of equal length, or approximately equal length. In particular embodiments, the section of continuous width constitutes at least 50, 60, 70, 80 or 90 % of the total length of the enclosed passage, and the tapered sections constitute at most 50, 40, 30, 20 or 10 % of the enclosed passage. In certain embodiments, the section of continuous width constitutes about 60, 70, 80 or 90 % of the enclosed passage. The Example below presents an embodiment in which the section of continuous width constitutes around 80 % of the total length of the channel. Importantly, in the enclosed passage there is a gap between the inlet and the first insert hole, and another gap between the final insert hole and the outlet (such that all insert holes are located between the inlet and outlet). That is to say, the inlet and outlet do not overlap with any insert holes, and nor are they directly adjacent to any insert holes. The first and last insert holes are located in the section of the enclosed passage of continuous width (i.e. no insert holes are located in the tapered sections of the enclosed passage, where these are present). The fast and last insert holes may be located at the ends of the section of the enclosed passage of continuous width. The gap between the inlet and the first insert hole, and the outlet and final insert hole, ensures that any swirling fluid patterns around the inlet and outlet do not reach the cells present on the tops of the islands, such that the fluid flow across all the islands is steady, uniform and laminar.

The gap between the inlet and the first insert hole (and correspondingly the gap between the final insert hole and the outlet) may be of any suitable length. For instance, the gap may be in the range 2-20 mm, e.g. 3-19 mm, 4-18 mm, 5-15 mm, 6-14 mm, 7-13 mm, 8- 12 mm or 9-10 mm.

Advantageously, the culture plate is rigid, that is to say made of a rigid material. That is to say the culture plate is preferably not flexible, and cannot be bent. The culture plate may be made of a polymer, particularly a plastic. Suitable plastics, particularly rigid plastics, are well known in the art. In some embodiments the culture plate is made of poly(methyl methacrylate) (PMMA). PMMA is also commonly known as acryclic.

Advantageously, the fluidic plate is rigid. That is to say the fluidic plate is preferably not flexible, and cannot be bent. The fluidic plate may be made of a polymer, particularly a plastic. Suitable plastics, particularly rigid plastics, are well known in the art. Preferably the fluidic plate is made of a transparent material. In some embodiments the culture plate is made of PMMA. In some embodiments both the culture plate and the fluidic plate are made of PMMA.

Advantageously, the island plate is flexible, that is to say made of a flexible material. That is to say that the island plate is not rigid and can be bent. The island plate may be made of a polymer, particularly a plastic. Suitable plastics, particularly soft plastics, are well known in the art. In some embodiments, the island plate is made of a silicone, preferably polydimethylsiloxane (PDMS). Alternatively, the island plate may be rigid, and in particular may be made of a rigid plastic. For example, the island plate may be made of polystyrene or polycarbonate, like a standard tissue culture plate.

Advantageously, the culture plate and fluidic plate are both rigid, as set out above, and the island plate is flexible, as set out above. This enables a straightforward assembly and disassembly of the cell culture configuration and the fluidic configuration. As detailed previously, for the purpose of cell culture, cells of interest are seeded into a well in the cell culture configuration, assembled from the cell culture plate and the island plate. As set out above, the base of the well is the top of a raised island. For analysis of the impact of fluid flow over the cells of interest to be analysed, it is necessary for the cells to adhere to the top of the raised island. To this end, the top of the raised island may be modified for cell adhesion thereto (i.e. the top of the raised island may be modified to cause, enable or improve the adhesion of cells thereto).

Any suitable modification known in the art can be performed, optionally selected based on the characteristics of the specific cells to be applied to the well and the material from which the island plate is made.

In some embodiments, the top of the raised island is tissue culture treated for cell adhesion thereto. Tissue culture treatment is well known in the art, and is achieved by exposing a surface to a plasma gas to make the surface more hydrophilic. In other embodiments, the top of the raised island is coated with a polypeptide or protein for cell adhesion. An example of a suitable polypeptide is polylysine. As is well known in the art, either poly-L-lysine or poly-D-lysine may be used.

Preferably, the protein used to coat the top of the raised island is an extracellular matrix (ECM) protein. Many ECM proteins are known in the art to be suitable for promotion of cell adhesion to a surface. Examples of suitable ECM proteins include laminin, collagen, fibronectin, vitronectin and tenascin, any of which may be used in the present invention. The ECM protein may be from any suitable animal, selected e.g. depending on the cells to be cultured. Particularly preferred is the use of fibronectin. An example of a suitable fibronectin protein is human fibronectin. The tops of the raised islands may be coated with an ECM protein by incubation with a solution of the chosen protein. The solution may be made up in any suitable solvent. Preferably the ECM protein solution is in a buffered solvent, e.g. phosphate-buffered saline (PBS). Other suitable solvents are known in the art. The ECM protein is at a sufficient concentration in the solution to coat the top of the island, e.g. in the range 5-50 pg/ml, e.g. 5-40, 5-30, 5-20, 5-15 or 5-10 pg/ml.

In some embodiments the system further comprises a base plate. The base plate is a plate used to form the base of the cell culture configuration and fluidic configuration. In both these configurations, when used the base plate is placed at the base (or bottom) of the configuration, below the island plate. The base plate is a flat plate upon which the island plate rests. When the culture and/or fluidic plate comprises a surround which surrounds the island plate in the assembled cell culture or fluidic configuration, the base plate extends across both the island plate and the surrounds of the culture or fluidic plate, that is to say both the island plate and the surround of the culture or fluidic plate rest upon the base plate. The base plate provides physical support and rigidity to all components when arranged in the cell culture configuration and the fluidic configuration. The base plate is preferably rigid, to provide a solid base to the cell culture and fluidic configurations. That is to say, the base is preferably made from a rigid material. The base may be made of a polymer, particularly a plastic, particularly a rigid plastic. In some embodiments the base is made of PMMA.

In some preferred embodiments, the base plate, culture plate and fluidic plate are all rigid (i.e. made of a rigid material), and the island plate is flexible (i.e. made of a flexible material). Preferably, the base plate, culture plate and fluidic plate are all made of the same material. In some preferred embodiments, the base plate, culture plate and fluidic plate are all made of PMMA. In some particular preferred embodiments, the base plate, culture plate and fluidic plate are all made of PMMA, and the island plate is made of PDMS.

The system of the invention may further comprise a lid for the culture plate. The lid can be placed on the cell culture configuration during cell culture, to protect the culture medium from contamination. The lid is commonly transparent, and may be made of a polymer, particularly a plastic. The lid is preferably rigid (i.e. made of a rigid material, particularly a rigid plastic). For example, the lid may be made of PMMA. The lid may be attached to the culture plate by any means. In some embodiments, the edges (that is to say, the lateral sides, i.e. the sides which are not the top or bottom) of the culture plate comprise a protruding lip, located between the top and bottom of the edges, upon which the lid rests when plated upon the culture plate. The protruding lip may alternatively be seen as a retracted offset.

The components of the system may be manufactured by any suitable means. For instance, the various plates may be made by moulding, 3D-printing, or any other suitable means for manufacturing the plates dependent on the material from which they are made. In some embodiments, as shown in the examples, the fluidic plate is constructed from two parts: a channel part and a top part. The channel part essentially forms the base of the fluidic plate, and comprises flow channels open at the top, i.e. non-enclosed passages for fluid, including the insert holes which run from the base of the open flow channel and open onto the base of the channel part. The channel part may also comprise an inlet and/or outlet for the flow channel, depending on where these will be located. The top part simply forms the top of the fluidic plate, and is preferably a flat or essentially flat plate which is attached to the top of the channel part, thereby enclosing the open passages by providing a top cover. The top part may comprise inlet and outlet holes for the flow channels, depending on where these are to be located. The top part and channel part are attached to each other to form the fluidic plate. The attachment may be by any suitable method known in the art, e.g. heat fusion (alternatively referred to as thermal fusion). Preferably, the fluidic plate is provided as a single component comprising flow channels as described above, but it may be provided in two separate parts, i.e. as a channel part and top part, for assembly into the fluidic plate.

The various components of the system may be attached to each other during assembly of the cell culture configuration and fluidic configuration by any suitable means. The components may simply rest on top of each other, particularly if the raised islands fit tightly into the through-holes and insert holes and thereby hold the components in place. Alternatively the components may be clipped or clamped together. In some embodiments, the island plate and/or the culture plate and fluidic plate comprises clips at the edges to enable direct attachment to the other component(s) as necessary.

The components of the system of the invention may be sterile. Sterilisation can be achieved by any method known in the art, e.g. heat, UV irradiation, chemically, etc.

In a second aspect, the invention provides a fluidic plate comprising at least one flow channel, each flow channel comprising an inlet, an enclosed passage and an outlet; wherein the inlet and outlet are located at either end of the enclosed passage, and each forms an opening between the enclosed passage and a side of the fluidic plate; wherein the enclosed passage of the at least one flow channel is tapered at both ends, and the inlet and outlet open into the tapered ends of the enclosed passage.

The fluidic plate of this aspect of the invention may be as the fluidic plate described above in the context of the system of the invention. However, the fluidic plate of this aspect of the invention need not contain insert holes within the flow channel(s). The fluidic plate of the invention may be for standalone use or use in an alternative system. When the fluidic plate is used outside of the system of the invention, and contains no insert holes, cells may be cultured directly within the flow channels before being exposed to flow conditions.

In a third aspect, the invention provides a kit. The kit is for cell culture and analysis, particularly for analysis of the response of cells to flow (e.g. to shear stress). The kit of the invention comprises the components of the system of the invention described above in the first aspect. Specifically, the kit comprises a culture plate as set out above, an island plate as set out above and a fluidic plate as set out above. As detailed above, preferably an intact fluidic plate is provided, but the kit may alternatively comprise a channel part and a top part for assembly into the fluidic plate.

In some embodiments, the kit further comprises a base plate as defined above. In some embodiments, the kit further comprises a lid as defined above. Preferably the kit comprises both the base plate and the lid. When the kit comprises a lid, the edges of the culture plate may comprise a protruding lip, as described above. The components of the kit may be provided sterile. Sterilisation of the components may be achieved by e.g. heat treatment, UV irradiation or chemically, e.g. using ethanol, or a combination of these means. The kit may further comprise a solution of an ECM protein for application to the top of the raised islands of the island plate, for promotion of cell adherence to the tops of the raised islands. The solution may be of any ECM protein, e.g. laminin, collagen, fibronectin, vitronectin or tenascin, as set out above. The solution may comprise a mixture of two or more ECM proteins. In some embodiments, the solution is a fibronectin solution. Any suitable fibronectin may be used (i.e. fibronectin from any animal), e.g. human fibronectin. The fibronectin used to coat the tops of the raised islands may be recombinant or native fibronectin isolated from cell culture. The fibronectin solution may be made up in any suitable solved. The solvent may be buffered, e.g. suitable solvents include PBS and CAPS saline solution. Fibronectin may be obtained commercially, e.g. from Merck (catalogue number F0556). The ECM protein (e.g. fibronectin) solution in the kit may be applied at any suitable concentration for application to the islands, e.g. in the range 5-50 pg/ml, e.g. 5-40, 5-30, 5- 20, 5-15 or 5-10 pg/ml. Alternatively, the solution may be provided at a higher concentration to be diluted prior to application to the islands.

The ECM protein solution is preferably sterile. Sterilisation may be achieved by any suitable method, e.g. microfiltration. The ECM protein solution may be provided in any suitable container, e.g. a tube.

In a fourth aspect, the invention provides a method of analysing the effect of shear stress on cells, the method comprising:

(i) providing, and optionally sterilising, a culture plate, an island plate and a fluidic plate, each as defined above in the first aspect;

(iv) removing the medium from the wells;

The culture plate, island plate and fluidic plate may be provided in a kit, as defined above. Step (i) preferably further comprises the provision of a base plate as defined above. Step (i) preferably further comprises the provision of a lid for the culture plate, as defined above. Preferably both a base plate and lid are provided. When a lid is provided, the edges of the culture plate may comprise a protruding lip, as defined above.

Step (i) comprises an optional sterilisation step. This need only be performed if the components are not sterile when provided. Means of sterilisation are detailed above and are routine for the skilled person.

The cell culture configuration is then assembled. This is achieved by placing the culture plate upon the island plate, such that the raised islands enter the through-holes of the culture plate resulting in the formation of wells. When a base plate is used in the method, the island plate is placed upon the base plate to form the cell culture configuration. As discussed above, any means may be used to secure the plates of the cell culture configuration together, e.g. clips or clamps.

The island plate may be provided with the tops of the islands pre-modified for cell adhesion thereto (as discussed above, the tops of the islands may be e.g. tissue culture treated or coated in a suitable ECM protein). If the tops of the islands are not pre-modified (i.e. are not modified prior to assembly of the cell culture configuration), following assembly of the cell culture configuration the tops of the islands may be modified for cell adhesion thereto (i.e. to promote cell adhesion) prior to seeding of cells into the wells. The tops of the islands may be modified by coating with a suitable polypeptide or protein, e.g. an ECM protein. Suitable ECM proteins for coating of the raised islands are detailed above. In some embodiments, the ECM protein is fibronectin. The tops of the raised islands may be coated with an ECM protein by incubating the tops of the raised islands in a solution of the desired protein. The protein solution may be made up in any suitable solvent as set out above, e.g. PBS. The protein solution may be at any suitable concentration, as set out above. The incubation may be performed at any suitable temperature, e.g. from 4-40°C, e.g. 4-37°C. In some embodiments the incubation is performed at room temperature, e.g. 18-25°C. The incubation may be for any suitable length of time, e.g. 10 min to 24 hours. About 1 hour may suffice.

Following application of the ECM protein to the tops of the raised islands, the ECM protein solution is removed from the wells. The tops of the wells may then be washed, e.g. with a suitable buffer such as PBS. The tops of the well may, for example, be washed once, twice or three times at this point.

In step (iii) the cells are seeded into the wells and then cultured therein. Any suitable cells may be used in this step, as desired for the purpose of the experiment. Most commonly endothelial cells are used, which may be either primary endothelial cells or cells of an endothelial cell line, though any other cell type may be used as desired. An example of a suitable endothelial cell line is the HLIVEC cell line (ATCC-CRL-1730). The cells used in this step may be human cells or derived from any other suitable animal. The cells may be native cells or may be modified to e.g. knock out or knock in a gene of interest, or otherwise modify the cells relative to native cells. Cells may be seeded at any appropriate concentration, as can be determined by the skilled person. For instance, cells may be seeded at a concentration of 1.8x10⁵ to 3x10⁶ cells/ml, as demonstrated in the Example. Any other suitable concentration may be used.

Following seeding the cells are cultured in the wells. The cells are cultured in medium (i.e. culture medium). The cells may be cultured in any suitable liquid culture medium, as known in the art, and under standard culture conditions. The cells may be cultured for any suitable period of time until they are considered ready for analysis, as determined by the skilled person.

If a lid for the culture plate is provided, this is placed on the culture plate at all times during the process, including during incubation of the wells with an ECM protein and during cell culture, to avoid contamination.

During culture, the cells may be contacted with an agent of interest, to determine the impact of that agent on the cells’ response to shear stress. The agent may be a small molecule, e.g. a putative drug, such as a drug candidate for treatment of vascular conditions. Commonly however the agent is an agent which modifies gene expression. Such an agent may be for knockdown of a target gene, e.g. an RNAi agent, particularly siRNA. When an RNAi agent such as siRNA is used the cells are transfected with the agent, using standard techniques in the art. Alternatively, if the cells have been modified to express a gene of interest under control of an inducible promoter, the cells may be contacted with the relevant inducer to induce expression of the gene of interest.

Following cell culture, the medium is removed from the wells. The cells may be washed at this point, e.g. with PBS. Thereafter the cell culture configuration is disassembled by removal of the culture plate from the island plate, and the fluidic configuration assembled by placing the fluidic plate upon the island plate, such that the raised islands enter the insert holes of the fluidic plate. As for the cell culture configuration, when a base plate is used, this is placed beneath the island plate. The constituent plates of the fluidic configuration are secured by any suitable means, e.g. clips or clamps.

Following assembly of the fluidic plate, fluid is flowed through the flow channels to expose the cells to shear stress. This is achieved by application of a continuous flow of fluid through the flow channels. The fluid enters the flow channel through the inlet, passes through the enclosed passage, over the cells adhered to the tops of the raised islands which form part of the base of the enclosed passage, and out through the outlet. By a continuous flow is meant that for the duration of the experiment the fluid enters the flow channel (through the inlet) at the same rate as it exits the flow channel (through the outlet) and thus an essentially continuous shear stress is applied to the cells. Any suitable fluid may be applied to the cells. The fluid should not of course be toxic to the cells, nor cause them to dissociate from the tops of the raised islands. A growth or culture medium may be used. The fluid may be applied to the cells for any suitable length of time to apply sufficient shear stress to them, e.g. 30 mins to 12 hours. The fluid may be pumped into the flow channels, e.g. using a peristaltic pump.

Following completion of the experiment, the flow of fluid through the flow channel is halted and the channel emptied of fluid. Thereafter the fluidic configuration is disassembled by removal of the island plate from the fluidic plate.

The cells adhered to the tops of the raised islands are then analysed to assess their response to the flow applied to them. Conveniently, the cell culture configuration may be reassembled at this point. The cells of interest can then be processed in the wells formed. The cells may be analysed by any means desired. The cells may be detached from the bases of the wells (i.e. the tops of the islands) and e.g. subjected to RNA extraction for RNA analysis (e.g. RT-PCR or RNASeq, etc.) or lysis for protein analysis (e.g. proteomics, immunoprecipitation or Western blot, etc.). Alternatively, the cells may be processed for microscopy, e.g. fluorescence microscopy, such as by immunofluorescence. Such techniques are standard in the art. To allow cells adhered to the tops of the islands to be examined by microscopy, the island plate may be transparent.

Thereby the impact of shear stress on the cells can be determined, and as relevant the effect of genetic manipulation of the cells’ shear stress response, or the effect of a small molecule of interest on shear stress response.

The present invention may be better understood by reference to the non-limiting examples and figures below. The components and parts referred to above have the following reference numbers in the examples and figures:

1 - cell culture configuration

2 - fluidic configuration

101 - culture plate

102 - island plate

103 - base plate

104 - through-holes

105 - raised islands

106 - fluidic plate

107 - channel part

108 - top part

109 - flow channels 110 - insert holes

111 - inlet

112 - outlet

113 - enclosed passage

114 - internal section of fluidic plate

115 - surround of fluidic plate

Figure Legends

Figure 1 shows (A) assembly of a cell culture configuration (1) from a culture plate (101), island plate (102) and base plate (103), exemplified using bulldog clips to hold the plates of the cell culture configuration (1) together; and (B) magnification of the culture plate

(101) and island plate (102) to better show through-holes (104) of the culture plate (101) and raised islands (105) of the island plate (102), along with dimensions of these plates and their features. The figures show CAD images of the plates and configuration. The figures show exemplary plates comprising 96 through-holes (104) and 96 raised islands (105).

Figure 2 shows photographs of, from left to right, a culture plate (101), island plate

(102) and assembled cell culture configuration (1). The photos include a 10 Euro-cent coin for scale.

Figure 3 shows (A) assembly of a fluidic configuration (2) from a fluidic plate (106), island plate (102) and base plate (103). In this example, the fluidic plate (106) is shown to be made from a channel part (107) and a top part (108). Again the assembly is exemplified using bulldog clips to hold the plates of the fluidic configuration (2) together. Magnification of the top part (108), channel part (107) (which form the fluidic plate (106)) and island plate (102) is shown (B) to better show the flow channels (109) and insert holes (110) in the fluidic plate (106) and the raised islands (105) of the island plate (102) along with dimensions of these plates and their features. As shown, the flow channels (109) comprise an inlet (111), outlet (112), and enclosed passage (113). In the example the inlet (109) and outlet (110) are located in the top part (108) of the fluidic plate (106). The fluidic plate is also shown to have an internal section (114) and surround (115). The surround (115) forms a cavity below the internal section (114) which houses the island plate (102) when the fluidic configuration (2) is assembled. The enclosed passages (113) are shown to have tapered ends. Figure 4 shows photographs of, from left to right, a fluidic plate (106), island plate (102) and assembled fluidic configuration (2). The photos include a 10 Euro-cent coin for scale.

Figure 5 shows the flow channels geometries used on the COMSOL software to perform simulations. Full channel size was simulated for this design.

Figure 6 shows (A) simulation of the velocity magnitude across the channel in the zx plane (longitudinal section); (B) simulation of the shear stress profile (N/m²) across the channel, in the zx plane (longitudinal section); (C) Simulation of the pressure profile (Pa) across the channel in the zx plane (longitudinal section); (D) simulation of the velocity magnitude across the channel in the yz plane (transverse section); (E) simulation of the shear stress profile (N/m²) across the channel in the yz plane (transverse section); and (F) simulation of the pressure profile (Pa) channel in the yz plane (transverse section). The simulations were performed for an initial velocity of 0.08m/s. Inlet and outlet are identified with a black arrow.

Figure 7 shows (A) simulation of the velocity magnitude (m/s) across the flow channel, in the xy plane at 10 pm distance above the bottom of the channel/well (z=10 pm); (B) simulation of the shear stress profile (N/m²) across the channel in the xy plane at 10 pm distance above the bottom of the channel/well (z=10 pm); (C) linear graph showing the variation of shear stress across the channel at 10 pm distance above the bottom of the channel/well (z=10 pm). Grey sguares represent the approximated location of the PDMS raised islands along the channel; (D) simulation of the pressure profile (Pa) across the channel in the xy plane at 10 pm distance above the bottom of the channel/well (z=10 pm). The inlet and outlet are identified with a black arrow.

Figure 8 shows (A) guantification of relative expression of KLF2 mRNA by RT-gPCR in HLIVECs exposed to static and flow (7 dyn/cm² of shear stress for 4 hours) conditions in the 96-well fluidic plate. **P<0.005 (unpaired Student's t-test). Error bars indicate standard deviation (n=3); (B) guantification of relative expression of KLF4 mRNA by RT-gPCR in HLIVECs exposed to static and flow (7 dyn/cm² of shear stress for 4 hours) conditions in the 96-well fluidic plate. ****P<0.0001 (unpaired Student's t-test). Error bars indicate standard deviation (n=3); (C) fluorescence images of HLIVECs exposed to static and flow (7 dyn/cm² of shear stress for 4 hours) conditions in the 96-well fluidic plate labelled for KLF4 (top images) and for nuclei (DAPI, bottom images); (D) guantification of KLF4 nuclear fluorescence intensity in HLIVECs exposed to static or flow (7 dyn/cm² of shear stress for 4 hours) conditions. ****P<0.0001 (Mann-Whitney test). Each point represents 1 cell. Box plot bars represent median, minimum and maximum values (n=2).

Figure 9 shows fluorescence images of control (siControl) aCatenin- and VE- cadherin-depleted (siCtnnal and siCdh5, respectively) HLIVECs under static (A) and flow (7 dyn/cm² of shear stress for 4 hours) conditions (B). HLIVECs were labelled for nuclei (DAPI), aCatenin (adherens junctions) and VE-cadherin (adherens junctions) (left-hand panels). Central panels show VE-cadherin labelling, and right-hand panels show aCatenin labelling. Scale bar: 20 pm.

Also shown (C) is quantification of relative expression of aCatenin mRNA by RT- qPCR in control (siControl) and aCatenin-depleted (siCtnnal) HLI ECs exposed to static and flow (7 dyn/cm² of shear stress for 4 hours) conditions. Expression levels are normalized to each control (siControl) condition. ****P<0.0001 (one-way ANOVA with Tukey correction for multiple comparisons). Error bars indicate standard deviation (n=3).

Also shown (D) is quantification of relative expression of VE-cadherin mRNA by RT- qPCR in control (siControl) and VE-cadherin-depleted (siCdh5) HLIVECs exposed to static and flow (7 dyn/cm² of shear stress for 4 hours) conditions. Expression levels are normalized to each siControl condition. ****P<0.0001 (one-way ANOVA with Tukey correction for multiple comparisons). Error bars indicate standard deviation (n=3).

Examples

Materials and Methods

Plate Design and Fabrication

The 96-well microfluidic plate system was designed using Autodesk (USA) AutoCAD® software. Dimensions were based on a regular cell culture 96-well plate to allow the usage of automated platforms to perform cell culture and transfection. The production of solid parts in poly(methyl methacrylate) (PM MA) was outsourced for higher resolution and higher refinement of finishing and bonding, to ZEG-MED, Poland. Manufacture was performed using a milling process. The polydimethylsiloxane (PDMS) parts were made by soft lithography from PMMA moulds.

The 96-well fluidic system features two functional configurations: the cell culture configuration (Figs. 1-2) and the fluidic configuration (Figs. 3-4). The cell culture configuration comprises two different parts, the island plate (96 raised islands) made of PDMS and the culture plate, made of PMMA. The island plate (127.74 x 85.40 mm) was produced using a PMMA mould, defining 2 heights: a first to define the flat base, with 3.5 mm height, and a second for the 6 mm x 6 mm islands, with 3 mm height.

For the culture plate, a PMMA plate (127.74 x 85.40 x 10.50 mm) was designed to enclose the 96 islands. 96 through-holes (6 x 6 x 10.5 mm) were drilled through the thickness of the plate (10.5 mm), into which the PDMS islands are inserted, creating a well 7.5 mm deep. The 96 culture wells formed from those through-holes are spaced 3 mm from each other. An important feature in this design is the fact that the top edges of the PMMA culture plate have a small retracted offset upon which a lid can rest, to maintain aseptic cell culture conditions. A PMMA base (153.29 x 102.48 x 5 mm) was also designed to give support to the cell culture and fluidic configurations (Figs. 1-2).

The fluidic plate is composed of two different PMMA parts (127.74 x 85.40 x 6.5 mm) that were bonded together by thermal diffusion bonding in order to create a single microfluidic flow part with 8 long channels each 0.5 mm deep. The top part (153.29 x 102.48 x 5 mm) is a PMMA plate that contains the inlets and outlets of the channels, each with 4 mm diameters. The lower part, or channel part (153.29 x 102.48 x 6.5 mm), comprises 8 channels (125.77 x 6 x 0.5 mm) with perpendicular insert holes for islands insertion. This design has several particularities. First, the flow channels start and end with a smaller width adjusted to the inlet/outlet inner diameter (4 mm), to create a stabilizing volume of media, prior to the PDMS islands. Second, below the 0.5 mm channel, the 96 insert holes (6 x 6 x 3 mm) create individualized boxes to house the PDMS islands, the tops of which align with the channel surface lower boundary, generating a linear flow channel. Third, a large cavity below the 96 insert holes was created to house the island plate, to improve the sealing of the assembly and minimize the leakage. This cavity was created by including a surround around the main internal section of the fluidic plate, which extends downwards by the height of the base of the island plate, so that it completely surrounds the island plate when the fluidic configuration is assembled. The same PMMA base plate is used as in the cell culture configuration to give mechanical support to the system, specifically to maintain pressure and avoid leakage.

Metallic clamps were used to hold all the constituent parts of the cell culture configuration and fluidic configuration tightly together (Figs. 3-4).

PDMS Preparation and Moulding

To prepare the PDMS component we used a PMMA mould designed using Autodesk AutoCAD® software. The mould is composed of two PMMA modules that were bonded by heat fusion in order to create a single mould. The first module is a smooth PMMA plate (153.29 x 102.48 x 5 mm) that gives rise to the top surface of the PDMS islands. The second module is a PMMA plate (153.29 x 102.48 x 6.5 mm) with a cavity, similar to the one described for the fluidic plate component. This cavity (127.74 x 85.40 x 6.5 mm) can be divided into two parts: the first part (3 mm high) contains 96 smaller cavities (6 x 6 x 3 mm) spaced 3 mm from each other which give rise to the PDMS islands. The second part (3.5 mm high) gives rise to the base of the island plate. To pour the PDMS the PMMA mould was turned upside down. A silicone elastomer was mixed with a curing agent (Sylgard™ 184 kit, Dow 101697) in a 10:1 ratio (w/w), followed by degassing in a vacuum chamber for 30 min at RT. Next, the PDMS was poured into the mould and another degassing step was performed to remove the bubbles formed during this process. The PDMS was cured by heating in an oven at 75°C for 2 h, and moulds kept level. After polymerization the PDMS was removed from the mould and ready to be used.

Device Assembly

Prior to assembling the device, all components were sterilized with 70 % EtOH and UV light in the laminar flow chamber, for 15 min. Then the cell culture configuration was assembled by attaching the PDMS island plate to the bottom of the culture plate, with the help metal clamps. Before seeding the cells, PDMS islands were incubated with 10 pg/ml fibronectin (diluted in PBS) for 1 h at RT, to ensure proper cell adhesion. After removal of fibronectin, the PDMS islands were washed twice with PBS followed by cell seeding and then transfection. For experiments under flow, the clamps were removed in order to replace the culture plate with the fluidic plate. The metal clamps were used again to close the system and an initial volume of media was introduced into the channels with the help of a syringe. During cell culture under flow, the PMMA base is used to fix all the components of the system in place and keep the system stable and tightly sealed, since the PDMS base is not rigid.

Computational Fluid Dynamics Simulation

To assess and better understand the fluidic behaviours in the perfused systems Computational Fluid Dynamics simulations were performed in COMSOL Multiphysics®, using the Fluid Flow module, specifically the single-phase fluid in the laminar flow regime, governed by the Navier-Stokes equation. The study was stationary, water defined as the equivalent fluid for cell culture media and the flow used was incompressible. The density and dynamic viscosity were defined from the material, in this case, water and the walls were defined as no slip wall. A CAD file with only 1 channel was imported to COMSOL (Fig. 5). Then a physics-controlled mesh using a Fine element size was built and the results of the simulation in terms of velocity, pressure and shear stress were analyzed for an initial velocity of 0.08 m/s. siRNA transfection

To silence the expression of genes of interest, a set of ON-TARGET human siRNAs against CTNNA1 (Dharmacon, GE Healthcare, J-010505-06), CDH5 (Dharmacon, GE Healthcare, J-003641-07) or untargeted control were used (Dharmacon, GE Healthcare, D-001810-01). Briefly, HLIVECs were seeded the day before the transfection at a concentration of 1.8 x 10⁵ cells/ml to reach 60-70 % confluence on the day of transfection. Then, cells were transfected with 25 nM siRNA using Dharmafect 1 reagent (Dharmacon, GE Healthcare) following the Dharmacon siRNA Transfection Protocol. The media was replaced 24 hours after transfection by fresh complete medium and cells were kept under culture conditions up until 72 h post-transfection and then processed for further experiments.

Flow on the 96-Well Fluidic System

As described above, to apply flow in the system, the culture plate is replaced with the fluidic plate. The channels are then filled with flow media - Leibovitz L15 media (LTI 21083-027, Life Technologies) supplemented with EGM-2 SingleQuotsTM (CC-4176, Lonza) and 1 % penicillin/streptomycin (#15140122, Gibco). The system is placed under culture conditions for 30 min to stabilize, and then the 96-well fluidic plate is connected to a peristaltic pump (Gilson Minipuls3), ensuring the continuous laminar flow during the duration of the experiment - 4 hours. The shear stress applied in the system was 0.7 Pa. After 4 hours, cells were fixed for IF or used to perform RNA extraction.

Cell Culture

Human umbilical vein endothelial cells (HUVECs) were routinely cultured following the manufacturer’s guidelines, in filter-cap T75 flasks Nunclon A surface treatment (VWR international, LLC) and cultured at 37°C and 5 % CO2 to ensure a stable environment for optimal cell growth. HUVECs (C2519A, Lonza) were cultured with complete medium EGM-2 Bulletkit (CC-3162, Lonza) supplemented with 1 % penicillin/streptomycin (#15140122, Gibco). When passaging cells for experiments, cells were washed twice in sterile PBS (137 mM NaCI, 2.7 mM KCI, 4.3 mM Na₂HPO₄, 1.47 mM KH₂PO₄, pH 7.4). Then, cells were incubated for 3-5 min in TrypLE™ Express (#12605-028, Gibco) at 37°C, 5 % CO₂. When 95 % of the cells detached, complete medium was added to each flask to inhibit the activity of the TrypLE™ Express and the cell suspension was transferred to a falcon tube. HUVECs were then centrifuged at 700 rpm for 5 min at room temperature and the pellet was resuspended in fresh complete medium. The cell concentration present in the suspension was determined using a Neubauer Cell Counting Chamber (Hirschmann EM Techcolor). The cells were then seeded on the desired culture system (ibidi, spotted slides, PDMS) at 1.8 x 10⁵ - 3 x 10⁶ cells/ml, depending on the experimental condition and placed in the incubator at 37°C, 5 % CO2. All experiments with HLIVECs were performed between passages 3 and 6.

Immunofluorescence

To perform immunofluorescence, HLIVECs were fixed in 1 % paraformaldehyde (PFA) supplemented with 1 M MgCh and 1 M CaCh (1 pL/2 mL) in PBS for 30 min at RT. Then, cells were washed with 1X PBS to remove the remaining PFA followed by blocking and permeabilization of cells with a blocking solution containing 3 % BSA in PBS-T (PBS with 0.1 % Triton X-100) for 30 min at RT. After, cells were incubated for 2 hours at RT with the appropriate primary antibodies diluted in the blocking solution (anti-VE-cadherin, R&D Systems - AF938, 1:50; anti-aCatenin, Sigma-Aldrich - C2081 , 1 :200; anti-KLF4, R&D Systems - AF3640, 1:200) and washed 3 x 15 min in PBS-T. Next, cells were incubated in blocking solution containing the appropriated secondary fluorophore-conjugated antibodies for 1 h at RT in the dark (donkey anti-goat Alexa 647, Thermo Fisher Scientific - A21447, 1:400; donkey anti-rabbit Alexa 568, Thermo Fisher Scientific - A10042, 1 :400) followed again by 3 washes of 15 min in PBS-T. Finally, cells were incubated with 1x DAPI (Molecular Probes by Life Technologies) diluted in PBS for 5 min in the dark, followed by 3 washes with PBS. PDMS islands were then mounted with a glass coverslip and using Mowiol DABCO (Sigma-Aldrich). Images were acquired using a confocal Laser Point-Scanning Microscope 880 (Zeiss) equipped with a Plan-Apochromat DIG 63x NA 1.40 oil objective and the Zen black software. To quantify the KLF4 nuclear intensity, 5 images per PDMS island were acquired. The mean fluorescence intensity was measured using the FIJI software. Briefly, the DAPI channel was used as reference to segment and select the nucleus as the region of interest (ROI) by setting a threshold. After, in the KLF4 channel, using the commands ROI to Manager and Analyze particles, the mean intensity fluorescence of KLF4 in static and flow conditions was measured, dictated by the ROIs defined in the DAPI channel.

RNA Extraction and cDNA Production

RNA was extracted from HUVECs seeded on the PDMS islands using Trizol (Alfagene). For each condition, cells from 3 PDMS islands were collected. Briefly, HUVECs were detached from the PDMS using TriplE™ Express (Thermo Fisher Scientific), as described in the cell culture section. Then, the cell suspension was transferred to an RNase-free Eppendorf tube and centrifuged for 5 min at 1000 rpm. After discarding the supernatant, the pellet was resuspended in Trizol and incubated for 10 min at RT. Afterwards, chloroform (Merck Millipore) was added. The tubes were shaken for 30 sec and incubated at RT for 5 min and centrifuged for 15 min. Afterwards, the upper aqueous phase was carefully transferred to a new tube. To precipitate the RNA, 1.5 pL Glycogen (Sigma) and 1/10 of the volume of the sample of 3 M sodium acetate were added to each tube. After vortexing, 1 volume of isopropanol (VWR) was added to each tube, following by vortexing, and tubes were then incubated 15 min at RT. After, tubes were centrifuged for 8 min and the supernatant was discarded. The pellets were washed with 1 mL of 70 % ethanol (VWR), the tubes were centrifuged for 5 min, and the supernatants were discarded. The pellets were allowed to dry at RT and then resuspended with 20 pL of RNase-free water (Sigma) and kept on ice, followed by RNA quantification using Thermo Scientific™ NanoDrop 2000. After quantification, samples were treated with RNase-free DNase I (Roche) for 20 min at 30°C. 1 volume of Phenol-Chloroform-isoamyl Alcohol mixture (Amresco) was added to the samples, to inactivate the DNase I and to purify the RNA. After centrifugation for 10 min, the upper aqueous phase was transferred for a new eppendorf, 1 volume of chloroform was added and tubes were centrifuged for 10 min. The upper aqueous phase was transferred to a new eppendorf and the precipitation and washing steps were repeated. After drying, the pellets were resuspended in 15 pL of RNase-free water, kept on ice and quantified using Thermo Scientific™ NanoDrop 2000. A fraction of the purified RNA (between 84 ng to 122 ng) was used to produce cDNA, using the High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems™), following the manufacturers’ protocol. The produced cDNA was stored at -20°C and used for RT-qPCR.

Real-Time Quantitative PCR (RT-qPCR)

To quantify the gene expression in static vs flow conditions in the 96-well fluidic plate, we performed RT-qPCR. In every RT-qPCR run, a standard curve was obtained for each primer pair alongside each sample, by mixing cDNA from all the conditions tested and then three different dilutions were made (1 :10, 1:25; 1:50). For each reaction a mix of 7 pL of Power SYBR® Green PCR Master Mix, 0.3 pL of primers (final concentration of 100 nM), 2 pL cDNA and 4.85 pL of RNAse-free water were prepared resulting in a final volume of 14 pL per well. The RT-qPCR reaction was performed in the Applied Biosystems VIIA 7 Real-Time PCR system using the standard protocol. The results were analyzed in the QuantStudio™ Real-time PCR Software (Applied Biosystems). The expression levels of each sample duplicate were then normalized to GAPDH, and the Livak Method (2-AACq) was used to calculate the relative changes in gene expression. The graphs were plotted using Graph Pad Prism 8 software.

Results Computational Fluid Dynamics

Analysis of velocity, shear stress and pressure profile were performed in a single representative channel of the 96-well fluidic plate. We accessed fluid dynamics at an initial velocity of 0.08 m/s. The plot of velocity magnitude in the xy plane, at 10 pm distance above the bottom of the channel/well (z= 10 pm) revealed a linear velocity profile with very small variations across the channel, with an increase in the velocity only near the inlet and outlet region, due to the smaller width of the channel in those regions (Fig. 7A). Additionally, transverse and longitudinal cross sections of velocity profile also show that the velocity has a minimal variation across most of the channel. The velocity reaches a maximum in the centre of the channel and a minimum value near the walls of the channel (Fig. 6A and D).

Similarly, shear stress profile in the xy plane, for a z=10 pm showed a very small variation across the channel, with higher values near the inlet and outlet due to the smaller width of the channel (Fig. 7B and C). In agreement, transverse and longitudinal cross sections showed no differences across the channel in terms of the shear stress profile (Fig. 6B and E). The pressure profile presented depicts the drop in the pressure from the inlet to the outlet as expected for the flow regime simulated (Fig. 4D; Fig. 6C and F). Thus, this simulation revealed that there are only minimal variations of velocity and shear stress profile across the channel area (in both X and Y), demonstrating high homogeneity per PDMS island along the same channel.

HUVECs Respond to Flow in the 96-Well Fluidic Plate

KLF2 and KLF4 are transcription factors sensitive to shear stress and that regulate downstream flow-dependent transcriptional responses in ECs. Several studies showed upregulation of KLF2 and KLF4 in cells subjected to laminar and pulsatile shear stress but not in disturbed flow conditions. In fact, KLF2 regulates around 46 % of high flow-responsive genes (Parmar et al., supra, Atkins and Jain, supra), most of them being atheroprotective genes under laminar flow. To validate the flow response of HUVECs in the 96-well fluidic system, we exposed ECs to a laminar and stable flow rate of 14 mL/min, corresponding to a shear stress of 7 dyn/cm² (0.7 Pa). This value was used on the basis of previous in vitro experiments, and which corresponds to an intermediate level of shear stress that ECs experience in veins in vivo (ranging between 1 dyn/cm² and 20 dyn/cm²). HUVECs were exposed to flow for 4 hours. To quantify EC flow response, KLF4 and KLF2 mRNA levels were assessed by RT-qPCR in HUVECs collected from individual channels in static or sheared conditions. For both genes, we observed a statistically significant increase in their expression under flow in comparison to static conditions (Fig. 8A and B). To further confirm these results, we quantified nuclear fluorescence intensity of KLF4 protein in similar conditions. We observed a significant increase in expression of KLF4 in these cells under flow, when compared to static conditions (Fig. 5C and D). The increased expression of KLFs not only demonstrates that ECs respond to flow in the 96-well fluidic system, but also strengths CFD simulations, indicative of a laminar and pulsatile flow regime. Thus, this 96- well fluidic system can be used to analyse general flow responses.

HUVECs Show High Levels of KD Efficiency in the 96-Well Fluidic Plate Next, we validated that our 96-well fluidic plate is compatible with efficient gene silencing. We targeted two different genes coding for important proteins forming adherens junctions in ECs: CTNNA1 and CDH5 that code for aCatenin and VE-cadherin, respectively. We selected these two targets due to the high selectivity of tools to quantitatively measure the efficiency of knock-down by immunofluorescence. In addition, adherens junctions have been reported as an important mechanosensor in ECs, thus allowing us to monitor changes in sensitivity of flow responses in knock-down cells. VE-cadherin is part of a mechanosensory complex together with PECAM1 and VEGFR2. Absence of VE-cadherin and PECAM1 affects EC-flow response by impairing the alignment of ECs under flow, but promoting polarity against the flow. We used previously validated small interference RNA (siRNA) against CTNNA1 and CD/75 to deplete aCatenin and VE-cadherin in HUVECS, respectively. We assessed levels of aCatenin and VE-cadherin 72 hours post-transfection in static and flow conditions. HUVECs transfected with siCTNNAI showed complete abrogation of aCatenin from adherens junctions and a strong reduction in the expression of VE-cadherin both in static and flow-stimulated cells. Similarly, siCDH5 transfected cells showed a dramatic reduction of both VE-cadherin and aCatenin expression in static and flow- stimulated cells (Fig. 9A and B). These observations were also confirmed and quantified by RT-qPCR, which clearly showed a statistically significant reduction of CTNNA1 and CDH5 in siCTNNAI and siCDH5 conditions, respectively, in both static and flow conditions (Fig. 9C and D). These results confirm that our 96-well fluidic plate is suitable to perform high- throughput siRNA-based screenings under laminar high flow conditions.

Claims

1. A system for cell culture and analysis comprising a fluidic plate, the fluidic plate comprising at least one flow channel, each flow channel comprising an inlet, an enclosed passage and an outlet; wherein the inlet and outlet are located at either end of the enclosed passage, and each forms an opening between the enclosed passage and a side of the fluidic plate; wherein the enclosed passage of the at least one flow channel is tapered at both ends, and the inlet and outlet open into the tapered ends of the enclosed passage.

2. The system of claim 1 , wherein the inlet of the at least one flow channel has a diameter greater than the width of the enclosed passage where the enclosed passage meets the inlet; and the outlet of the at least one flow channel has a diameter greater than the width of the enclosed passage where the enclosed passage meets the outlet

3. The system of claim 1 or 2, wherein the fluidic plate further comprises at least one insert hole which forms an opening between the enclosed passage and the bottom of the fluidic plate, wherein the at least one insert hole is located between the inlet and the outlet; the system further comprising:

4. The system of claim 3, wherein the culture plate and/or the fluidic plate comprises an internal section and a surround, wherein the internal section comprises the one or more through-holes or insert holes, and the surround extends below the base of the internal section thereby forming a cavity, wherein the cavity is dimensioned to house the island plate when the cell culture configuration or fluidic configuration is assembled.

5. The system of claim 3 or 4, further comprising a base plate which forms the base of the cell culture configuration and the fluidic configuration.

6. The system of any one of claims 3 to 5, further comprising a lid for the culture plate, optionally wherein the edge of the culture plate comprises a protruding lip upon which the lid rests when placed upon the culture plate.

7. The system of any one of claims 3 to 6, wherein the culture plate, the fluidic plate and, when present, the base plate are rigid, and the island plate is flexible.

8. The system of claim 7, wherein the culture plate, the fluidic plate and/or the base plate are made of poly(methyl methacrylate) (PMMA), preferably wherein the culture plate, the fluidic plate and the base plate are all made of PMMA.

9. The system of claim 7 or 8, wherein the island plate is made of a silicone.

10. The system of claim 9, wherein the silicone is polydimethylsiloxane (PDMS).

11. The system of any one of claims 3 to 10, wherein the top of the at least one raised island is modified for cell adhesion thereto.

12. The system of claim 11 , wherein the top of the at least one raised island is coated with an extracellular matrix (ECM) protein, preferably fibronectin.

13. The system of any one of claims 3 to 12, wherein:

(i) the culture plate comprises 96 through-holes, the island plate comprises 96 raised islands and the fluidic plate comprises:

(a) 8 flow channels, each comprising 12 insert holes, or

(b) 12 flow channels, each comprising 8 insert holes; or

(ii) the culture plate comprises 384 through-holes, the island plate comprises 384 raised islands and the fluidic plate comprises:

(a) 16 flow channels, each comprising 24 insert holes, or (b) 24 flow channels, each comprising 16 insert holes.

14. A kit comprising:

(i) a fluidic plate as defined in any one of claims 1 to 13;

(ii) a culture plate as defined in any one of claims 3 to 13; and

(iii) an island plate as defined in any one of claims 3 to 13.

15. The kit of claim 14, further comprising:

(i) a base plate as defined in any one of claims 5 to 13; and/or

(ii) a lid as defined in claim 6, optionally wherein the edge of the culture plate is also as defined in claim 6.

16. The kit of claim 14 or 15, further comprising a solution of an ECM protein for application to the top of the at least one raised island of the island plate, preferably wherein the ECM protein is fibronectin.

17. The kit of any one of claims 14 to 16, wherein the components are sterile.

18. A method of analysing the effect of shear stress on cells, comprising:

(i) providing, and optionally sterilising, a fluidic plate as defined in any one of claims 1 to 13, a culture plate as defined in any one of claims 3 to 13 and an island plate as defined in any one of claims 3 to 13;

(iv) removing the medium from the wells;

19. The method of claim 18, wherein a base plate as defined in any one of claims 5 to 13 is provided and used in assembly of the cell culture configuration and the fluidic configuration; and/or a lid as defined in claim 6 is provided and used during cell culture, and the edge of the culture plate is optionally as defined in claim 6.

20. The method of claim 18 or 19, further comprising modifying the tops of the raised islands for cell adhesion prior to seeding the cells.

21. The method of claim 20, wherein the tops of the raised islands are modified for cell adhesion by application of an ECM protein, preferably fibronectin.

22. The method of any one of claims 18 to 21 , wherein during the cell culture the cells are contacted with an agent which modifies gene expression, optionally an siRNA agent.

23. The method of any one of claims 18 to 22, wherein a peristaltic pump is used to apply the flow of fluid through the flow channel.