Devices for capturing tissues and / or multicellular objects

Abstract

A microfluidic device comprising a microfluidic module, the microfluidic module comprising a plurality of fluid paths between an input and an output, and a plurality of limiters, each limiter located on each of the plurality of fluid paths, and each limiter configured to capture or at least restrict the movement of one or more objects, e.g., one or more tissues and / or multicellular objects, in the fluid introduced along each fluid path in its respective sensing region, the fluid module further comprising at least one further fluid path between an input and a further output, the at least one further fluid path being in fluid communication with the plurality of limiters, the further fluid path comprising a delivery portion between the input and the plurality of limiters for enabling the delivery of further fluid to the captured and / or restricted one or more objects, and an output portion for enabling the removal of further fluid from the further output, the device further comprising an electronic sensing module, the electronic sensing module comprising at least one sensing element arranged to detect one or more signals from each of the sensing regions of the plurality of limiters.

Description

Technical Field

[0001] Field The present disclosure relates to microfluidic devices, and in particular to devices for capturing and testing a plurality of objects, for example one or more tissues and / or multicellular objects. For example, methods of using said devices to determine the quality and / or function of cells, tissues or objects are provided.

Background Art

[0002] Background Type 1 diabetes mellitus (T1D) is an autoimmune disease affecting 400,000 people in the UK (JDRF Editors (2022)). Its symptoms are characterized by the loss of functional beta cells, which are the majority of cells located within the pancreatic islets, a group of cells in the pancreas. Each islet contains approximately 1,500 cells, 74% of which are beta cells. These beta cells normally secrete insulin in response to glucose stimulation, but this function is lost in T1D, resulting in poorly controlled and / or high blood glucose levels. Treatment with synthetic insulin injections can help control blood glucose levels and is central to treatment, but there is no feedback control, and hypoglycemia is a common side effect of insulin therapy. Repeated episodes of hypoglycemia can lead to hypoglycemic impaired consciousness, which prevents individuals from recognizing low blood glucose levels, potentially resulting in significantly higher morbidity and mortality. Transplanting islets from deceased donors to treat T1D has been shown to be effective in improving glucose regulation. Transplantation can reduce the frequency of severe hypoglycemia and is an effective procedure for restoring awareness of hypoglycemia. In fact, in a small number of cases, this procedure can restore glycemic control by achieving insulin independence (Forbes et al. (2015)). However, patient outcomes depend on several factors, including the quality and quantity of transplanted islets. Currently, the availability of donor pancreases with islets is the main limiting factor; in the UK, approximately 100 were available in 2019, but many were unsuitable for transplantation or had insufficient quality and quantity of islets (Cornateanu et al. (2021)). This situation is further complicated by the fact that typically 2-3 donor islets are needed per organ transplant patient to obtain a significant effect on glycemic control.

[0003] Islet count, sample purity, and islet viability are all critical parameters for meeting clinical release criteria before transplantation (Brooks et al. (2013), Benomar et al. (2018)). In the UK, islet viability must be over 70% for preparation transplantation. Overestimating islet viability can lead to the transplantation of a relatively large number of unviable and inadequately sized islets, while underestimating it can lead to the discarding of otherwise viable islets. These criteria protect organ transplant patients and ensure that only pancreases with a sufficient number of healthy islets are transplanted.

[0004] However, this has resulted in a decrease in the clinical conversion rate, which fell to 28% in 2019 (Bunnett J & Counter C (2019)). Nevertheless, according to the collaborative islet transplant registry, approximately 70% of islet transplant patients were insulin-independent one year after transplantation, but this decreased to approximately 40% at five years after transplantation (CITR Coordinating Centre (2015)). Therefore, in the case of T1D, insulin independence is achievable, but the proportion tends to decrease in the first few years after transplantation, demonstrating that the quality and quantity of islets are important for favorable long-term outcomes. In summary, the decline in post-transplant clinical outcomes and the low percentage of donor pancreases that meet release criteria point to the urgent need for objective islet assessment aimed at improving the number and quality of islets for transplantation.

[0005] Currently, membrane integrity staining with fluorescein diacetate and propidium iodide (Barnett et al. (2004)) is the preferred method for assessing viability. While this approach is simple and easy to apply, it has the problem of frequent overestimation of viability due to the subjective interpretation of results (Boyd et al. (2008)). There is accumulating evidence that inflamed and dying islets can negatively affect the entire islet graft, potentially impairing the function of originally healthy islets. Therefore, developing a robust and more objective method for real-time assessment of islet viability before transplantation is an urgent priority.

[0006] Measuring the electrophysiological response of β-cells in pancreatic islets to glucose is an attractive alternative assessment because the electrical response produced by β-cells is a necessary step in the healthy glucose-stimulated insulin secretion (GSIS) pathway, and therefore represents an objective, label-free method for determining viability. Determining the electrical activity of β-cells typically required conventional electrophysiological methods such as single-cell and / or whole-cell voltage clamping techniques or intracellular electrodes (Pfeiffer et al. (2011), Dufer (2012)). Such techniques are very time-consuming and require specialized single-cell manipulation and interpretation. On the other hand, extracellular recording using aspirated electrodes and microelectrode arrays (MEAs) makes it possible to detect the electrical activity of pancreatic islets with minimal tissue microscopy. In fact, the electrical activity of intact mouse and human pancreatic islets has been demonstrated in single and multiple islets, and this approach has been shown to be a viable method for assessing islet viability, since the absence or abnormality of electrical activity is an indicator of loss of GSIS function (Pfeiffer et al. (2011), Schoenecker et al. (2014, 2015)).

[0007] Therefore, while previous studies have shown the electrophysiological responses of isolated pancreatic islets, these approaches require specialized equipment, knowledge, and skills to manually manipulate individual islets and interpret the results. [Overview of the project] [Problems that the invention aims to solve]

[0008] One of the purposes of this disclosure is to avoid and / or mitigate at least one of the aforementioned drawbacks. [Means for solving the problem]

[0009] overview In a first embodiment, a microfluidic device is provided, comprising a microfluidic module, the microfluidic module comprising a plurality of fluid paths between an input and an output, and a plurality of limiters, each limiter located on each of the plurality of fluid paths, and each limiter being configured to capture or at least restrict the movement of one or more objects, e.g., one or more tissues and / or multicellular objects, in the fluid introduced along each fluid path in its respective sensing region, the microfluidic module further comprising at least one further fluid path between an input and a further output, the at least one further fluid path being in fluid communication with the plurality of limiters, the further fluid path comprising a delivery portion between the input and the plurality of limiters for enabling the delivery of further fluid to the captured and / or restricted one or more objects, and an output portion for enabling the removal of further fluid from the further output. The device may further comprise an electronic sensing module comprising at least one sensing element arranged to detect one or more signals from each of the sensing regions of the plurality of limiters.

[0010] A sensing module may comprise one or more passive electrodes for measuring potential. A sensing module may include one or more stimulating electrodes configured to generate an electronic stimulus in an object to produce a response that can be measured by the passive electrodes. A sensing module may comprise one or more stimulating elements and one or more sensing elements. The stimulating and sensing elements may be positioned to stimulate and detect electrical activity in a sensing region. A sensing module may comprise one or more stimulating electrodes and one or more sensing electrodes. A sensing module may form part of a sensing and stimulating module configured to provide a stimulus in a sensing region and measure the response to the stimulus.

[0011] The sensing module may comprise one or more stimulating elements configured to generate an electronic stimulus, and one or more sensing elements for measuring the detected signal, optionally an electrical potential. The stimulating elements may be configured to generate an electronic stimulus, and the sensing elements may be configured to detect a response to the stimulus.

[0012] The object may include at least one of organoids, cell spheroids, tissue spheroids, and / or pancreatic islets. The object may also include cell objects and / or tissue objects.

[0013] The signal may include extracellular signals. The device may comprise multiple microfluidic modules. The device may comprise multiple microfluidic modules and corresponding multiple electronic sensing modules.

[0014] The object may include tissue spheroids derived from the pancreas. The tissue and / or multicellular object may include cells having at least one functional, structural, or biological characteristic of an organ, for example.

[0015] The additional fluid path may at least partially overlap with the multiple fluid paths in the delivery portion, and the output portion is spatially separated from the multiple fluid paths.

[0016] The output portion of the further fluid path may be located downstream of the delivery portion and the plurality of limiting portions.

[0017] The further fluid path may include a path for flushing and / or removing at least fluid and material from the device via the further output.

[0018] The first channel may include a fluid and / or object delivery portion. The first channel may include a fluid and / or object flushing portion. Multiple limiting portions may be provided in or on the fluid and / or object delivery portion.

[0019] Multiple limiting sections may be provided adjacent to and in fluid communication with at least one further fluid path. At least one further fluid path may be arranged to expose the captured and / or limited object to further fluid. The delivery section may be a shared delivery section or may form part of the first channel. The output may have a common output.

[0020] The device may comprise a plurality of channels. The plurality of channels comprises a first channel between the input and the further output, the first channel defining the further fluid path between the input and the further output, and the plurality of channels further comprises a second channel coupled to the plurality of limiters and the output, the second channel being coupled to the first channel via the plurality of limiters such that at least a portion of the first channel and at least a portion of the second channel define the plurality of fluid paths between the input and the output.

[0021] The first channel and the second channel may be sized to allow fluid flow of the object. The restrictor may have an opening between the first channel and the second channel that is sized to prevent the object from passing through.

[0022] The input may comprise an inlet. The output may comprise an outlet. A further output may comprise a further outlet. Fluid may flow from the input to the further output through the further fluid channel at a higher flow rate when the restriction is substantially occluded than when the restriction is not substantially occluded. The at least one restriction may form part of a trap for capturing one or more objects, for example one or more tissues and / or objects. The objects may be cellular objects and / or tissue objects. The restriction may be dimensioned to allow fluid to flow through the restriction while preventing objects from passing through the restriction.

[0023] The at least one sensing element may comprise an electrode. The at least one sensing element may comprise a passive high-impedance electrode for measuring an electric field potential relative to a passive or grounded low-impedance reference electrode. The at least one sensing element may be provided in a sensing region. The device may comprise a hybrid microfluidic and microelectronic device.

[0024] The microelectrode array may comprise one or more stimulation electrodes for stimulating one or more sensing regions and / or captured objects and / or one or more objects within the sensing region. The one or more stimulation electrodes may comprise a pair of stimulation electrodes for stimulating the sensing region, optionally an object within the sensing region. The one or more stimulation electrodes may comprise an electrode pair utilizing a low-impedance reference electrode.

[0025] The detection module may comprise at least one passive sensing electrode element and, for example, at least one stimulation electrode and a reference electrode. The stimulation electrode and the reference electrode may form a stimulation electrode pair. The device may comprise a plurality of stimulation electrode pairs. The at least one stimulation and reference electrode pair may be arranged on a first side of the detection region, and the at least one sensing electrode may be arranged on a second side of the detection region, such that the detection region is provided between the at least one stimulation electrode pair and the at least one sensing electrode. One of the stimulation electrode pair may be provided on a first side of the one or more detection regions, and the other of the stimulation electrode pair may be provided on another side of the detection region, such that one or more detection regions are provided between the stimulation electrode pair. The device may comprise a stimulation electrode shared between one or more detection regions. At least one stimulation electrode may be provided on a first side, a reference electrode pair may be arranged on the first side of the detection region, and at least one sensing electrode may be arranged on the second side of the detection region, such that the detection region is provided between the at least one stimulation electrode pair and the at least one sensing electrode. The at least one sensing electrode may be configured to detect a response in an object to an electrical stimulus delivered from the stimulation electrode pair. One or more electrodes or sensing elements may be provided above or below a microfluidic device layer.

[0026] The detection module may comprise at least one passive sensing electrode element and, for example, at least one stimulation electrode and a reference electrode. The stimulation electrode and the reference electrode may form one or more electrode pairs. The electrode pairs may be arranged on both sides of the detection region such that the detection region is provided between the stimulation electrode and the sensing electrode. The at least one sensing electrode may be configured to detect a response in an object to an electrical stimulus delivered from the stimulation electrode.

[0027] The plurality of restriction portions may include 5 or more, optionally at least 10, optionally at least 15, optionally at least 60, optionally at least 100 restriction portions.

[0028] The multiple limiting sections may be positioned in the overlapping portions of the multiple fluid paths and the further fluid paths to enable the continuous delivery of objects to the multiple limiting sections, such that when one limiting section is blocked by an object, further objects in the fluid proceed to subsequent limiting sections and / or toward the further output.

[0029] Multiple fluid paths and further fluid paths may be formed by channels of dimensions such that one or more objects can flow through them.

[0030] The at least one sensing element may optionally be positioned on a layer below the plurality of restricting portions such that at least partially restricted and / or captured objects come into contact with the at least one sensing element.

[0031] At least one detection element may comprise multiple detection elements, each comprising one or more detection elements for each limiting section and at least one reference electrode.

[0032] The sensing element may be configured to detect electrophysiological signals and / or signals and / or biological signals in response to the electrophysiological activity of the object. The electrophysiological activity or other activity of the object may respond to chemical substances and / or chemical stimuli applied in the input. The electrophysiological activity or other activity of the object may respond to chemical substances and / or chemical stimuli applied in the input and flushed through the system. The electrophysiological activity or other activity of the object may respond, for example, to electrical stimuli applied to the object from one or more stimulating elements or electrodes.

[0033] The at least one sensing element may be provided as part of an electrode array aligned with and / or located near the at least one limiting portion. The electrode array may be aligned to contact the one or more objects. The electrode array may be aligned to make optimal contact with the one or more objects. The at least one sensing element may comprise one or more sensing electrodes or recording electrodes. The sensing electrodes may be passive electrodes. The sensing electrodes may include an exposed portion that contacts the object. The exposed portion may have a diameter in the range of 10 to 100 microns, and optionally 20 to 60 microns. The diameter of the exposed portion may depend on the material used.

[0034] The sensing module may comprise multiple conductive tracks. The conductive tracks may be coupled, for example, to corresponding portions, such as electrode pads, or form part of multiple electrodes. The tracks may lead to the corresponding electrode pads. The tracks may be conductive. The tracks may be electrically isolated from the fluid paths and / or channels of the microfluidic device. During use, the tracks may be electrically isolated from the fluid contents of the microfluidic device. The pads and / or tracks may contain conductive materials, such as titanium or gold deposits. The tracks may be insulated by insulating materials, such as silicon oxide.

[0035] At least one pair of stimulating electrodes may be provided as part of an electrode array, aligned with and / or adjacent to at least one limiting element and passive recording electrode. The electrode array may be substantially planar.

[0036] The device may further include a reference sensing element. The device may further include a reference sensing electrode, such as a passive electrode. The reference sensing element may be provided in or across at least a portion of the plurality of fluid paths and / or at least a portion of the further fluid paths. The reference sensing element may form part of a pair of stimulating electrodes. The reference sensing element may be provided together with the stimulating electrodes. The sensing module may comprise one or more active elements and one or more passive elements.

[0037] The reference sensing element may act as a reference sensing element for at least one, and optionally more, sensing elements. At least a portion of the reference sensing element may be located in, or overlapping with, multiple fluid paths and / or further fluid paths. Multiple sensing electrodes may be aligned with multiple limiting elements. Multiple sensing electrodes may be arranged in a substantially linear arrangement. The spacing between subsequent sensing electrodes may correspond to the spacing between multiple limiting elements. The sensing elements may be arranged such that each limiting element is substantially located between the corresponding sensing element and the reference element. The exposed portion of the reference electrode may be at least a portion, optionally all or all, of the electrode pad that provides a passive low impedance voltage reference. The exposed portion of the reference electrode may comprise a passive low impedance component.

[0038] Each of these limiting sections may have an opening with a cross-sectional area that is at least 50%, 60%, 70%, 80%, 90%, or 95% smaller than the diameter of the one or more objects in question.

[0039] The plurality of fluid paths and the at least one further fluid path may be formed by channels having a cross-sectional area at least 50%, 60%, 70%, 80%, 90%, or 95% larger than the diameter of the one or more objects in question.

[0040] The object may have dimensions, for example, a width and / or height ranging from 150 microns to 300 microns.

[0041] Multiple limiting sections may have a width smaller than the width of the object in question. The limiting sections may have a width of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the width of the object in question.

[0042] Multiple limiting sections may have widths between 10 and 75 microns, and optionally between 20 and 40 microns. The channel width may be in the range of 300 to 1000 microns, optionally between 400 and 500 microns, and optionally 450 microns. The channel height may be in the range of 200 to 1000 microns, optionally between 200 and 300 microns, and optionally 250 microns.

[0043] Multiple fluid paths and at least one further fluid path may be formed by channels having a width at least 50%, 60%, 70%, 80%, 90%, or 95% greater than the width of one or more objects. Multiple fluid paths and at least one further fluid path may be formed by channels having a height at least 50%, 60%, 70%, 80%, 90%, or 95% greater than the height of one or more objects.

[0044] The width and / or dimensions of the object in question may include the average or typical width and / or dimensions of such object.

[0045] The multiple fluid paths and the further fluid paths may be formed by multiple channels. At least one of the widths and / or heights of at least one channel may be selected from a range that increases or decreases the fluid velocity.

[0046] The microfluidic device may further include a chip holder and / or amplifier connected to computer reading software. The microfluidic device may be used to evaluate the electrical activity, viability, function, and / or response of cells or tissue objects to chemical and / or physical stimuli.

[0047] The device may further include processing resources. The processing resources are configured to receive sensor signals and / or data representing the sensor signals from the at least one sensing element, and to process the data and / or signals to determine at least one of electrical activity, viability, function and / or response to stimuli.

[0048] The data and / or signals may be processed to determine the percentage of viable tissue and / or material in the sample. Determining at least one of electrical activity, viability, function, and / or response to stimulation may include generating data representing electrical activity, function, and / or response to stimulation, and storing such data.

[0049] The processor may be configured to determine at least one of the following: a) A measure of electrical activity in response to further fluid and / or direct electrical stimulation, optionally the further fluid comprising chemical substances and / or drugs, such as a glucose solution, and further optionally the electrical stimulation being provided by one or more stimulating elements or electrodes, and at least one of the following further: b) Some viable and / or functional tissues and / or multicellular objects in the sample, c) Diagnosing and / or indicating viability based on detected electrical activity in multiple limiting and / or sensing regions, for example, the diagnosis and / or indicating viability may optionally be based on electrical activity in response to exposure of the object to further substances, including chemicals and / or drugs, such as glucose, and / or electrical stimuli. The chemicals and / or drugs may include the chemicals and / or drugs being tested. The response may be in response to metabolic stimuli such as glucose and / or electrical stimuli.

[0050] A second aspect provides a method for providing one or more objects of interest to a device according to the first aspect. The method includes the step of introducing one or more objects in a fluid into the input of the device, the fluid initially flowing through the device until the one or more objects reach a plurality of limiters and are captured and / or at least restricted by the plurality of limiters.

[0051] A third aspect provides a method for analyzing a plurality of objects, such as a plurality of tissues and / or multicellular objects, using the device according to the first aspect. The method includes the step of providing the plurality of objects in a fluid to the input of the microfluidic device, the fluid initially flowing through the device along the plurality of fluid paths until the plurality of objects reach the plurality of limiting parts and are captured and / or at least restricted by the limiting parts, and the method further includes the step of detecting at least one sensor signal for the plurality of objects using the at least one sensing element.

[0052] The method may further include the step of delivering a further fluid through the input to the plurality of objects trapped or at least restricted within the plurality of restrictors via the at least one further fluid path. The method may further optionally include the step of applying electrical stimulation via one or more pairs of electrodes.

[0053] At least one sensor signal may represent or indicate the biological activity of multiple objects. At least one sensor signal may include one or more first sensor signals detected before the delivery of further solutions and / or electrical stimuli, and one or more further sensor signals detected after the delivery of further solutions and / or electrical stimuli. One or more first sensor signals may represent or indicate the reference electrophysiological activity of multiple objects. Further signals may represent or indicate the electrophysiological activity of multiple objects. At least one sensor signal may be detected for each limiting section, optionally by a sensing element provided in the limiting section, and optionally between the sensing element in each limiting section and the reference electrode. With respect to the reference electrode, at least one sensor signal may be detected for each limiting section.

[0054] The one or more objects in question may originate from the pancreas, for example, the islets of Langerhans.

[0055] One or more objects may originate from other commonly available spheroidal objects such as cardiac spheroids, nerve organoids, and other excitatory object organoids.

[0056] One or more objects may move to and / or beyond at least one limiting element by gravity, bulk flow, and / or capillary action.

[0057] The further fluid may include a washing solution for removing any solution and / or object via the further output. The further fluid may include a glucose solution. The further fluid may include one or more drugs and / or objects or cell activity inhibitors. The further fluid may include one or more molecules for labeling the object. The further fluid may have a higher concentration than the fluid containing the multiple objects.

[0058] The method may further include the step of isolating the output electrical signals from the trapped object for the plurality of limiting parts of the microfluidic device. The method may also include the step of isolating the electrical signals for at least 15, optionally 30, and optionally 60 traps.

[0059] The method may further include processing the sensor signal and / or data representing the sensor signal to evaluate at least one of the electrical activity, viability, function and / or response to stimuli of a cell or object.

[0060] According to a fourth aspect, an apparatus is provided comprising the device and processing resources of the first aspect. The processing resources may correspond to the processor of the first aspect. The processing resources may be configured to receive a sensor signal and / or data representing the sensor signal from the at least one sensing element, and to process the data and / or signal to determine at least one of electrical activity, viability, function and / or response to a stimulus.

[0061] The data and / or signals may be processed to determine the percentage of viable tissues and / or cells and / or objects in the sample. Determining at least one of electrical activity, viability, function, and / or response to stimulation may include generating data representing electrical activity, function, and / or response to stimulation, and storing such data.

[0062] The processor may be configured to determine at least one of the following: a) a measure of electrical activity in response to a further fluid, optionally including a chemical and / or drug, such as a glucose solution; b) several viable and / or functional tissues and / or multicellular objects in the sample; and c) making a diagnosis and / or indication of viability based on the detected electrical activity in a plurality of limiting and / or sensing regions. For example, such diagnosis and / or indication of viability may optionally be based on the electrical activity in response to exposure of the object to further substances, including chemical and / or drugs, such as glucose, and / or direct electrical stimulation of the object.

[0063] The device may further include a display and user input. In a fifth embodiment, a microfluidic / microelectronic / multichannel device is provided, wherein at least one of the microfluidic modules is (a) A microfluidic module comprising a first channel for receiving fluid, the first channel comprising an input and an output, and at least 10 traps positioned between the input and the output and for capturing multiple tissue / organoid samples introduced into the first channel through the input, the traps comprising a limiting portion, the limiting portion being sized to allow fluid to flow through the limiting portion but not to allow one or more tissue / organoids to pass through the limiting portion, the traps further comprising at least 10 electrodes for detecting electrophysiological signals of one or more tissues captured in the traps, and at least one of the microfluidic modules further, (b) A second channel is provided for receiving fluid from the first channel after the restriction is blocked by one or more tissues / organoids, thereby preventing further fluid flow through the restriction. The second channel may also be used to remove fluid and / or material after analysis.

[0064] This disclosure is generally described in the context of pancreatic islets, but this should not be construed as limiting. The devices of this disclosure may provide any type of tissue (e.g., cardiac or nervous tissue) in which the electrical response generated by multiple active cells and / or objects therein allows for the evaluation of its viability and / or function. In one embodiment, one or more tissues may be derived from the pancreas. In a preferred embodiment, one or more tissues derived from pancreatic islets contain, for example, several thousand β-cells. While pancreatic islets are provided herein as exemplary cells and / or objects, this disclosure is not intended to be limited to such cells and / or objects, as excitability changes in membrane potential can be used to evaluate the viability and / or function of various types of excitable tissues.

[0065] Membrane potential refers to the voltage or potential difference across the cell membrane. This potential difference arises from hydrophobic membranes that separate charges and act as both capacitors and resistors for the movement of charged ions across them. The plasma membrane ensures the structural integrity of the cell and physically separates the intracellular compartment from the extracellular peripheral region. Potential gradients exist across the cell membrane due to substantial differences in ionic composition between the intracellular and extracellular compartments, selective permeability to specific ion species, and the insulating physical properties of the phospholipid bilayer, including the cell membrane. Under steady state, the intracellular compartment of a cell is more negatively charged with respect to the extracellular environment (with respect to potassium ions), resulting in a resting membrane potential ranging from -10mV to -80mV depending on the cell type. In excitable tissues such as pancreatic islets (including those derived from stem cells), muscle, and neurons, rapid changes in membrane potential (e.g., action potentials) can be induced by electrical and / or chemical signals that alter the permeability of ions through ion channels. The action potential has a duration of several milliseconds and an amplitude exceeding 100 mV.

[0066] The electrophysiological state and / or response of a cell typically refers to the electrical properties or activity of the cell, which are reflected in the changes in membrane potential described above. The charge underlying the electrical state or activity of a cell is, for example, sodium (Na). +) ions, chloride (Cl - ) ions, potassium (K + ) ions and calcium (Ca 2+ These are dependent ions such as ions. The flow of ions into and out of cells is regulated by ion channels, and the movement of charged ions across the cell membrane can generate an action potential scale voltage difference (approximately 100 mV). Electrodes, microelectrodes, or microelectrode arrays can be used externally to detect the synchronous cellular electrical activity of a single or large number of cells by measuring and / or detecting extracellular local electric field potentials, for example, which are actually very small potential changes (5 μV to 100 μV). Such approaches are clinically used to measure brain potentials (electroencephalogram: EEG), electrocardiogram (ECG), and electromyogram (Eelectromyogram: MG), in which many cells are synchronously active.

[0067] Accordingly, in alternative embodiments, the disclosure may relate to objects, organoids, or tissues derived from other organs, and the evaluation of the function and / or response of the entire object by changes in the extracellular local electric field potential is relevant. Without limiting to the examples provided herein, alternative organs from which the object tissues of the method may be derived may include, for example, the heart, brain, intestines, liver, kidneys, gallbladder, stomach, or skin and endothelial tissue, as well as objects derived from stem cells from certain organs. Objects (or cells) derived from organs, and tissues for use in accordance with the disclosure, may include cultured cell objects, particularly cultured mammalian materials, for testing the viability and / or function of such cultured objects. In some embodiments, the tissues of the disclosure may include groups or clusters of cells that can be supplied to a microfluidic device. For example, such tissues may include organoids (e.g., brain organoids, cardiac spheroids) or objects derived from embryonic stem cells or induced pluripotent stem cells (iPSCs). In some embodiments, the tissues may include one or more clusters of cells or objects, or spheroids of cells or objects. Therefore, those skilled in the art will recognize that the dimensions of the first channel, the second channel, the trap, and / or the limiter can be appropriately adapted depending on the dimensions of the tissue, cell cluster, or group of cells provided to the microfluidic device for testing. In particular, at least the first channel and / or the trap should be sized such that at least one tissue object can fit within the dimensions of the first channel and / or the trap.

[0068] The input serves as an inlet path through which fluid can be supplied to the microfluidic module. In a preferred embodiment, a fluid, such as a liquid containing one or more tissue objects (e.g., cell culture medium, saline, buffer, etc.), is supplied to the microfluidic module through the input. The input may comprise an input chamber through which fluid, such as a fluid reservoir, is supplied to the channels of the microfluidic module. The fluid supplied to the input flows through the channels, preferably by gravity and capillary-driven flow. The use of gravity or capillary flow ensures that the tissue objects are handled in the gentlest possible manner and distinguishes it from prior art systems that may use pumps or the like to drive or aspirate fluid and objects through the microfluidic device. The microfluidic module also comprises outputs (such as output channels and / or wells / reservoirs) through which the fluid that has flowed through the channels can be collected. In some embodiments, the microfluidic modules of a microfluidic device are connected to the same input. In some embodiments, the microfluidic modules of a microfluidic device are connected to the same output. The fluid flow through the device delivers one or more objects to traps, which occlude the fluid flow through each limiting section.

[0069] In one embodiment, the trap has a cross-sectional area at least 50%, 60%, 70%, 80%, 90%, or 95% larger than the cross-sectional area of ​​one or more objects to be tested. In one embodiment, the trap area has a cross-sectional area at least 50% larger than the cross-sectional area of ​​one or more objects leading to the limiter. In a preferred embodiment, the maximum diameter of the cross-sectional area of ​​the trap and / or limiter may be 10 μm to 60 μm. The trap is adjacent to a limiter of a size such that tissue clusters or objects cannot pass through the limiter. The limiter preferably has a cross-sectional area such that it prevents one or more objects from passing through and captures them, so that the limiter is blocked to prevent or minimize the flow of fluid through the limiter after the capture of an object. For example, it is known in the art that clusters such as mammalian object tissue, pancreatic islets, and cardiac spheroids typically have a diameter of 100 μm to 300 μm. Therefore, in one embodiment, the maximum diameter of the limiting cross-sectional area may be 5%, 10%, 20%, 30%, 40%, 50%, or 60% of the diameter of the object provided to the microfluidic device. In one embodiment, the maximum diameter of the limiting cross-sectional area may be 40 μm ± 10 μm. In a further embodiment, the dimensions of the channel may include a width of 450 μm ± 50 μm and / or a height of 450 μm ± 50 μm.

[0070] After the fluid flow through the limiting section is blocked by one or more objects provided to the microfluidic device, any fluid flowing through the device is directed primarily toward and through the second channel. It should be noted that the fluid may flow through the second channel before and after the blocking of each limiting section by one or more of the structural objects. The second channel is also referred to as a bypass or bypass channel throughout this disclosure. In one embodiment, the cross-sectional area of ​​the second channel may be at least 50%, 60%, 70%, 80%, 90%, or 95% larger than the cross-sectional area of ​​one or more structures. In some embodiments, the cross-sectional area of ​​the second channel may include a cross-sectional area at least 50% larger than the diameter of the object.

[0071] In one embodiment, the microfluidic device may include a single microfluidic module. In such an example, when the limiting section is closed, the fluid flows through the second channel toward the output. In other embodiments, the microfluidic device may comprise multiple microfluidic modules, thereby providing multiple traps and limiting sections within each device. In a multi-module configuration, the second channels of each module may be connected to form a continuous second channel leading to the same output. In some embodiments, the direction of fluid flow through the second channel associated with an object trap is such that the fluid flow direction is toward the second channel only when each limiting section is closed and the fluid overflows from the object trap into the second channel.

[0072] A multi-module configuration of microfluidic devices equipped with microelectronic substrates provides a high-throughput means. This high-throughput means allows for the simultaneous measurement of electrophysiological activity from multiple tissues, enabling the determination of the percentage of functional tissue among them. This feature offers significant advantages, such as when it is necessary to rapidly evaluate the quality of a large number of pancreatic islets before transplantation. Importantly, the devices of this disclosure overcome the limitations of existing technologies, which often require complex instrument setups, have low throughput, and / or damage to objects during operation.

[0073] In further embodiments, the dimensions of the first channel, object or spheroid trap, limiter, and / or second channel may be modified to accommodate various tissue types and / or dimensions by adding adapters or stoppers that can limit and / or increase the cross-sectional area. In one embodiment, the microfluidic module includes adapters or stoppers for modifying the cross-sectional area of ​​the first channel and / or second channel at a defined point within a specified range. In a particular embodiment, the channel dimensions may include a diameter, height, or width in the range of 200 μm to 500 μm. In an alternative embodiment, the object trap and / or limiter may include adapters or stoppers for modifying the diameter, height, or width of the channel depending on the type of object provided to the microfluidic device.

[0074] Each microfluidic module of the present disclosure may comprise at least one electrode for evaluating the electrophysiological state and / or response of an object to a stimulus. The microfluidic device of the present disclosure comprises at least 10 electrodes, which are preferably located in each object trap so that the electrical activity of the object can be detected. Typically, a reference electrode is required to obtain a background signal or reference signal of the electric field. In a preferred embodiment, the microfluidic module comprises an integrated ground reference electrode. The ends of each electrode reach into the microchannel so that measurements can be obtained from within the channel. In one embodiment, each module of the microfluidic device comprises at least 10 recording electrodes. In a preferred embodiment, the electrodes of the present disclosure comprise microelectrodes. In some embodiments, one or more electrodes may form a microelectrode array. Microelectrode arrays are commonly used in the art to measure the electrical activity of an object. In a preferred embodiment, the electrodes comprise gold electrodes and / or titanium electrodes.

[0075] However, in a multi-module configuration, not all modules need to have electrodes. In some embodiments, one or more modules of the microfluidic device do not include electrodes. One or more modules lacking electrodes can be used as a control group in which the tissue used in the assay can be microscopically visualized, or they can be used for biochemical assays, such as gene expression, compound detection, or viability assays using optical detection (e.g., absorbance or fluorescence). In such applications, one or more modules may be access points through which tissue can be isolated for analysis, and through which a solution immersing the tissue (e.g., for a secretion assay) can be collected without disturbing the tissue. To enable visualization of one or more tissues through the microfluidic device, in some embodiments, one or more modules may have a polymer coverslip or glass coverslip bottom so that the object can be directly visualized through the device. If electrical information is required, transparent electrodes can be printed on these coverslips.

[0076] Microfluidic devices may be fabricated from, for example, plastic, glass, silicone, or other materials. In a preferred embodiment, the channel of the microfluidic device comprises transparent polydimethylsiloxane (PDMS). In the examples disclosed herein, transparent PDMS is used for the channel and borosilicate glass is used as the substrate for the titanium (Ti) electrode (insulated by silicon dioxide deposition), but any transparent substrate or molded plastic known in the art to be suitable for fabricating microfluidic devices may be used.

[0077] One or more electrodes of the device can be prepared by a number of methods known in the art. In one embodiment, the microfluidic device according to the disclosure may have one or more electrodes prepared by a printing method, for example, by plasma bonding or pressure fixing.

[0078] The devices of this disclosure may further include a chip holder and / or amplifier connected to computer reading software. Commercially available amplifiers may be used, but are not limited to, a Model 3600 amplifier which can function as a multi-channel extracellular differential AC amplifier. For example, the amplifier may typically have, per channel, at least 16 channel capabilities, at least 10 gain settings, at least 5 low-pass filters in the range of 50 Hz to 50 kHz, at least 5 low-pass filters in the range of 0.1 Hz to 600 Hz, and a notch filter (e.g., 50 Hz or 60 Hz). In some cases, the signal may be pre-amplified by a headstage typically located proximal to the electrodes, and the recording is acquired by the amplifier.

[0079] Those skilled in the art will understand that the microfluidic devices of this disclosure may have a variety of applications. In one embodiment, a microfluidic device may be used to evaluate the electrical activity, viability, function, and / or response of an object to a stimulus. The stimulus may include any substance that induces a change in the cellular activity of a population, such as glucose, ions, hormones, ligands, electrical stimulation, or drugs. In a preferred embodiment, the stimulus provided to the microfluidic device includes glucose.

[0080] A sixth aspect provides a method for adding one or more tissues to a microfluidic device disclosed herein in order to measure the electrical activity of one or more tissues. The method includes the step of providing one or more tissues in a fluid to the input of the device, the fluid initially flowing through the device until one or more tissues reach a trap and block the fluid flow through a limiter. The blockage of the fluid flow through the limiter facilitates the fluid flow through a second channel to the output of the microfluidic device.

[0081] A seventh aspect provides a method for analyzing an object using the microfluidic device of the Disclosure. The method is (i) The method includes the step of supplying one or more tissues in a fluid to the input of a microfluidic device, the fluid initially flowing through the device until one or more objects reach a trap and block the fluid flow through the limiting portion, and the method further, (ii) A step of measuring a reference electrophysiological activity for one or more tissues through one or more electrodes, (iii) The step of delivering a test solution to one or more tissues via an input which would make an object somewhat electrically active, wherein the substrate may specifically stimulate or block electrical activity from particular cells, for example, high glucose concentration to pancreatic islets, and the method further, (iv) A step comprising measuring the electrophysiological activity of one or more tissues through one or more electrodes in response to a test solution.

[0082] In one embodiment, the method may include a plurality of test solutions, and steps (iii) and (iv) are repeated, with an optional step (v) comprising supplying a washing solution through an input after each solution in order to measure the electrophysiological activity of one or more tissues in response to each test solution.

[0083] In some embodiments, after the delivery of tissue to the trap and occlusion of the restriction section, the fluid of step (i) may be removed by suction of the fluid from the output before the test solution of step (iii) is added. In alternative embodiments, the output may initially be blocked by a stopper, which is released by gravity and capillary-driven forces to remove the solution of step (i) before the test solution of step (iii) is added.

[0084] In one embodiment, one or more tissues (islets of Langerhans) are derived from the pancreas. For optimal object viability, one or more tissues of the method disclosed herein are supplied to and / or maintained in solution within the microfluidic device in a solution. In a preferred embodiment, one or more tissues are supplied to the microfluidic device in a solution such as a commercially available cell culture medium suitable for the tissue type. Once the one or more tissues are supplied to the microfluidic device, the tissue objects move along a first channel toward a trap to block the fluid flow through a limiting section and to enable measurement of the electrophysiological activity of the objects through electrodes. In one embodiment, one or more tissues move through the first channel by gravity and capillary-driven fluid flow.

[0085] In one embodiment, the culture medium and / or test solution may contain glucose. In one embodiment, the concentration of glucose in the solution may be 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, or 20 mM. In an alternative embodiment, the concentration of glucose in the solution may include any concentration physiologically relevant to the state or tissue type under consideration.

[0086] In one application, the microfluidic devices and / or methods described herein may be used as a drug discovery platform or for safety screening of compounds. In addition, or alternatively, the culture medium and / or test solution may contain one or more agents to evaluate changes in the electrophysiological response of tissue. One or more agents may be agents that enhance cell viability for therapeutic purposes and / or agents that alter the electrophysiological response of tissue. One or more agents may include ion channel agonists or ion channel antagonists. For example, one or more agents may include, for example, tolbutamide, chlorpropamide, or gliclazide. Alternatively, the electrophysiological activity of an object may be manipulated by altering the ion concentration of the extracellular medium. In one embodiment, the test solution contains higher concentrations of components (e.g., ions, glucose, other nutrients) including the culture medium. Optionally, and depending on the circumstances, one or more agents may include hormones that halt the electrical activity in the object, for example, insulin in the case of pancreatic islets.

[0087] Methods according to this disclosure enable rapid and accurate label-free assessment of tissue viability and / or function. Alternatively, the method may include additional steps of isolating one or more tissues from a first channel and / or a fluid from a microfluidic device for further analysis. One or more tissues may be isolated for further analysis of one or more tissues, for example, to evaluate the electrical activity, viability, function, and / or response to stimuli of the object. Further analysis may include various techniques known in the art, such as transcriptome analysis or fluorescence microscopy. In some embodiments, the test solution may contain one or more molecules for labeling the object, and the labeled tissue is isolated for further analysis. For example, one or more molecules for labeling the object may include any commercially available fluorescent probes or dyes for staining cellular components.

[0088] Features in one embodiment may also be provided as features in another embodiment. For example, features of a method may be provided as features of a device or apparatus, and vice versa.

[0089] Simple explanation Various aspects of the present invention are described herein by reference only as examples. [Brief explanation of the drawing]

[0090] [Figure 1] Figure 1 is a top view showing a multi-channel device microfluidic channel having multiple traps according to one embodiment. [Figure 2] Figure 2 is a magnified view of the device shown in Figure 1. [Figure 3] Figure 3 is a further enlarged view of the device in Figure 2, showing indicators of the flow direction and where objects are likely to be captured. [Figure 4] Figure 4 shows a top view and a cross-sectional view illustrating further single-trap devices as background examples. [Figure 5a] Figure 5(a) is a top view showing a single-trap device. [Figure 5b] Figure 5(b) shows a multi-trap device according to the embodiment. [Figure 5c] Figure 5(c) is a photograph of a microfluidic module of a device bonded to a glass microelectrode array. [Figure 5d] Figure 5(d) shows an example of a multi-channel microfluidic chip and electrode array mounted on a holding module. [Figure 5e] Figure 5(e) is a further photograph showing a microfluidic module of the device bonded to a glass microelectrode array. [Figure 6a] Figure 6(a) shows further diagrams and micrographs illustrating the multichannel device. [Figure 6b] Figure 6(b) shows further diagrams and micrographs illustrating the multichannel device. [Figure 6c] Figure 6(c) shows further diagrams and micrographs illustrating the multichannel device. [Figure 6d] Figure 6(d) shows further diagrams and micrographs illustrating the multichannel device. [Figure 6e] Figure 6(e) is a composite micrograph showing pancreatic islets trapped within the device. [Figure 7a] Figure 7(a) shows a detection element module of a device according to one embodiment. [Figure 7b] Figure 7(b) shows a detection element module of a device according to one embodiment. [Figure 8a] Figure 8(a) shows a sensing element module of the device according to a further embodiment. [Figure 8b] Figure 8(b) shows a sensing element module of the device according to a further embodiment. [Figure 9] Figure 9 shows an apparatus according to one embodiment. [Figure 10a] Figure 10(a) shows a plot of results obtained using the device. [Figure 10b] Figure 10(b) shows a plot of results obtained using the device. [Figure 10c] Figure 10(c) shows a plot of results obtained using the device. [Figure 10d] Figure 10(d) shows a plot of the results obtained using the device. [Figure 11a] Figure 11(a) is a further diagram showing the device in Figure 1. [Figure 11b] Figure 11(b) is a further diagram showing the device in Figure 1. [Figure 12] Figure 12 is a further diagram showing the device in Figure 1. [Figure 13] Figure 13 is a top view showing a device according to one embodiment. [Figure 14a]Figure 14 is a top view showing a device having a stimulating electrode according to one embodiment. [Figure 14b] Figure 14 is a top view showing a device having a stimulating electrode according to one embodiment. [Modes for carrying out the invention]

[0091] Detailed explanation This disclosure relates to a device. According to embodiments, the device may be used to determine the quality, maturity, or developmental state of islet Langerhans tissue by electrical activity measurement. The device may also be deployed in a similar manner to measure the electrical activity of nerve cells or organoids of similar dimensions (100 μm to 300 μm in diameter) and any other organoids, including but not limited to neural stem cells and cardiac spheroids.

[0092] Electrode-based approaches to assess the viability of newly isolated pancreatic islets have generally been considered impractical. For example, in "Microelectrode Array based Functional Testing of Pancreatic Islet Cells" (Alassaf et al. (2020)), extracellular recording of pancreatic islets using a flat MEA (microelectrode array) platform with conventional MEA chips (Figure 1A) (Alassaf et al. (2020)) was shown to be impractical, requiring proper contact and adhesion between the islets and electrodes for recording electrical activity. Given that pancreatic islets are large multicellular spheroids, the limited contact area with the recording electrodes on a flat MEA hinders MEA recording for functional assessment of pancreatic islets. In contrast to the device described herein, Alassaf et al.'s pancreatic islets were subsequently dissociated into single cells on the array and cultured for several days. Known approaches using imaging label the islets, making them unsuitable for transplantation. Alternative known approaches may damage the islets, making them unsuitable for transplantation. Hereafter, the terms organoid, spheroid, islet, and object are used as examples of tissue or tissue culture. In this sense, organoids may refer to clusters of cells derived from embryonic stem cells, induced pluripotent stem cells, or organ-specific adult stem cells developed in culture to form an identifiable structure possessing some or all of the anatomical and physiological / functional characteristics of a normally developed organ. They typically consist of thousands of cells. These include, but are not limited to, stem cell-derived islets, neural stem cell-derived brain organoids, cardiac spheroids, kidneys, ovaries, intestines, and spleens. These tissues or objects are approximately 100 to 300 microns in diameter. Primary organoids, on the other hand, may refer to tissues derived from donor islets as well as donor material of the same scale, such as muscle and brain biopsies. The devices described below can measure excitation in excitable tissues, and the methods for determining functionality are best applied to excitable endocrine tissues such as islets, brain, and muscle.

[0093] There is an unmet need in the art for the development of novel devices and methods that can efficiently identify viable and functional clusters of cells, including islets for transplantation, without chemical labeling or biochemical manipulation. Rapid identification of viable islets is crucial, as transplant outcomes are often closely related to the number of islets and their viability. In the context of islet transplantation, for example, the transplantation process is a multi-step procedure involving pancreatic procurement, tissue dissection, islet purification, islet culture, and islet transplantation into the liver of the organ transplant recipient via the hepatic portal vein. At each stage, islets are lost (primarily due to ischemia). Furthermore, it is estimated that less than 50% of islets from a single pancreas are isolated (approximately 500,000 islets), and less than 50% of these engraft in the liver. Therefore, there is an urgent need for novel devices and methods that can rapidly assess the quality of islets without adversely affecting their viability and function. This disclosure is based on an approach that eliminates the need for complex manipulation of pancreatic islets (or other objects mentioned, including organoids and cell clusters) and enables testing of multiple islet responses (or other objects mentioned) quickly enough to give predictive estimates of batch viability without requiring chemical manipulation or labeling.

[0094] It will be understood that all living cells possess bioelectrical activity, and generally, many cells can respond to chemical or physical stimuli that can increase or decrease their bioelectrical activity. For example, cardiomyocytes / cardiomyocyte organoids / stem cell-derived cardiomyocytes may be stimulated with isoprenaline and their electrical activity measured. Clusters of viable cells may generate extracellular coordinated electrical signals at rest and in response to such pharmacological or electrical stimulation (pacing), while clusters of non-viable cells will not generate coherent electrical signals or any electrical signals at all; for this reason, clusters of viable cells and clusters of non-viable cells will be distinguished. Therefore, this device can also be applied to stem cell-derived neural objects and other groups of nerve cells and other organoids.

[0095] Figure 1 is a top view of a device having multiple traps according to one embodiment. It will be understood that the device 100 has a microfluidic module and an electronic sensing module, and that Figure 1 shows only the microfluidic module. The electronic sensing module will be described in more detail, for example, with reference to Figure 7 and subsequent figures. The device 100 is configured to capture multiple objects, in particular biological objects such as tissues and / or multicellular objects. Objects may include cellular objects or tissue objects. In this embodiment, each trap includes a restricting section for restricting or capturing tissue or organoids in a cell culture. In the following description, the device is described as capturing cell spheroids, but it will be understood that the device may be used to capture objects derived from cultures or tissue cultures, e.g., organoids, spheroids, e.g., object spheroids, tissue spheroids, and pancreatic islets. Such objects are suspended in a fluid and transported through the device's channels by the fluid flow. The device may be for capturing and testing excitable tissues and / or multicellular objects to test bioelectrical activity as a surrogate indicator of tissue viability.

[0096] The microfluidic module has an input 110, also referred to as a common input or shared input, and an output 111, also referred to as a common output or shared output. The microfluidic module also has a further output 113, which is separate from the shared output 111. The microfluidic arrangement has multiple fluid paths between the input 110 and the shared output 111. In the embodiment shown in Figure 1, there are 11 defined fluid paths between the input 110 and the shared output 111, with each fluid path passing through a limiting section. As described above, each limiting section is configured to capture one or more tissues and / or multicellular objects in the fluid introduced into the device via the input 110 and transported along the respective fluid path of the limiting section.

[0097] In addition to the multiple fluid paths defined between the input 110 and the shared output 111 via the limiting section, an additional fluid path is defined between the input 110 and an additional fluid output 113. The additional fluid path is in fluid communication with the multiple limiting sections but does not pass through them. The additional fluid path has a delivery section between the input 110 and the multiple limiting sections 108 that enables the delivery of fluid to the limiting sections. The additional fluid path has an output section via the additional output 113 that enables the removal of fluid from the device. The microfluidic arrangement is such that the multiple limiting sections are provided adjacent to and in fluid communication with the additional fluid path. Thus, the additional fluid path is arranged to expose captured and / or restricted objects to the additional fluid. The additional fluid path may be called a fluid delivery path and / or a cleaning path. The multiple fluid paths may be called an object delivery path.

[0098] In the embodiment shown in Figure 1, the device has a first channel 101 defined between an input 110 and a further output 113. The first channel incorporates an input portion 102 (also called the input channel portion), an intermediate portion 115, and a bypass portion 104 (also called the bypass channel portion). The input portion 102 is coupled to the intermediate portion 115 at a first coupling 113, and the intermediate portion 115 is coupled to the bypass portion 104 at a second coupling 112, so that the intermediate portion 115 is located between the first coupling 113 and the second coupling 112. The device has a second channel 106 (also called the exit channel) coupled to the first channel 101 via a limiting portion. The second channel has a first portion 106a and a second portion 106b. The second channel 106 is coupled to the first channel along the first portion 106a via a plurality of limiting portions 108. In particular, the multiple limiting sections 108 provided along the intermediate portion 115 of the first channel connect the intermediate portion 115 to the first portion 106a of the second channel 106. The input portion 102 and the intermediate portion 115 together may form a fluid and / or object delivery portion. The bypass portion 104 may form a fluid and / or object flushing portion. Therefore, the multiple limiting sections may be provided in or on the fluid and / or object delivery portion.

[0099] It will be understood that the first and second channels of the device in Figure 1 are sized to allow fluid flow of an object. Therefore, in this embodiment, the first channel is sized to allow flow of the object in question, and the limiting portion is sized to prevent flow of the object in question. In this embodiment, the object in question is a cell spheroid. Generally, the object in question may be sized in the range of 150 to 300 microns. In this embodiment, the first and second channels have a width of 450 microns and a height of 250 microns, and the limiting portion is 40 microns. It will be understood that alternative dimensions may be used in other embodiments. In particular, these design parameters may be selected depending on the object in question. The channels are formed in a PDMS layer with dimensions of 3 cm × 3 cm × 0.5 cm.

[0100] In the embodiment shown in Figure 1, further fluid paths are defined by a first channel 101 between the input 110 and a further output 113. At least a portion of the first channel 101 and at least a portion of the second channel 102 define multiple fluid paths between the input 110 and the output 111. In particular, in the embodiment shown in Figure 1, the multiple fluid paths are defined between the input 110 and the output 111 via the input channel portion 102, the intermediate portion 115 of the first channel, the first portion 106a of the second channel, and the second portion 106b of the second channel.

[0101] In the embodiment shown in Figure 1, the delivery portion of the additional fluid path lies between the input 110 and the junction 112 between the intermediate portion 115 of the first channel output and the bypass portion 104 of the first channel. The additional fluid path at least partially overlaps with the multiple fluid paths between the input 110 and point 112. It will be understood that the bypass portion 104 of the additional fluid path is spatially isolated from the multiple fluid paths. Thus, the additional fluid path provides a path for flushing and / or removing fluid and / or material and / or debris from the device via the additional output 113.

[0102] In the embodiment shown in Figure 1, the input channel portion 102 is a common or shared input channel portion for delivering fluid to each of the multiple traps. Also, as is clear from Figure 1, the bypass channel portion 104 is a common output channel portion for the multiple traps, and the outlet channel provides a common output channel for the multiple traps. In the embodiment shown in Figure 1, the device 100 has 11 traps, but it will be understood that in other embodiments, a different number of traps may be provided.

[0103] The first channel 102 has an input channel portion 102 and an intermediate portion 115 between the fluid input 110 and the second coupling portion 112. The input may be an inlet port, and / or the output may be an outlet port. Multiple traps 108 are arranged between the input and output of the first channel 102, particularly along the intermediate portion 105. As shown in Figure 1, each of the multiple traps 108 is in fluid communication with the first channel portion 102, the bypass channel portion 104, and the outlet channel 106. In the embodiment of Figure 1, the multiple traps are arranged in series such that, during use, the fluid flowing through the input channel 102 is supplied to each of the cell spheroids and captured sequentially, and the multiple traps have a common input channel.

[0104] The direction of the fluid flow can be defined as starting from the input 110. In the direction of the fluid flow between the input and the further output 113, it will be understood that the output portion of the further fluid path is downstream from the delivery portion and the multiple limiting portions.

[0105] In the above-described embodiment of Figure 1, input 111 corresponds to the inlet port of the device, and output corresponds to the outlet of the device. In some embodiments, it will be understood that fluid paths may be defined between inputs and outputs that do not correspond to inlets / outlets. For example, multiple fluid paths may be defined between an input defined at a point along the input channel 102 (e.g., at a point substantially at the coupling 113) and a shared output defined along the output channel 106. Similarly, further fluid paths may be defined between the same input along the input channel 102 and further outputs defined along the bypass channel portion 104. In such embodiments, multiple inlets may be coupled to defined inputs, and / or multiple outlets may be coupled to the shared output and / or further outputs. In such embodiments, the further fluid paths have a delivery portion that overlaps with the multiple fluid paths and an output portion that is spatially separated from the multiple fluid paths.

[0106] As described above, in the embodiment of Figure 1, the channels define fluid paths through the device. In particular, for each trap, there is a corresponding fluid path from the fluid input 110 to the shared output 111, and the limiter is the limiter of that corresponding fluid path. Thus, multiple fluid paths are defined between the fluid input 110 and the shared output 111 via multiple limiters. In addition, in the embodiment of Figure 1, as described above, the first channel 101 defines further fluid paths that bypass the limiters and pass through the device. The fluid path of each trap delivers objects to the limiter of the trap. Thus, objects are prevented from flowing along their fluid paths by the limiters. The further fluid paths are in fluid communication with each of the limiters and can be used to deliver additional fluid to the captured object to enable fluid exchange and / or further fluid delivery. Each fluid path is defined between the input 110 and the shared output 111 via its respective trap. Therefore, each fluid path at least partially overlaps with a further fluid path between the input 110 and the bypass channel portion 104, and in particular, the overlapping portions correspond to at least a part of the input portion 102 and the intermediate portion 115.

[0107] Figure 2 shows two of the multiple traps in Figure 1 in more detail. Figure 2 shows the first trap 108a and the second trap 108b. As shown in Figure 2, the first trap 108a has a limiting section 114a. The second trap 108b also has a limiting section 114b. It will be understood that each of the multiple traps of device 100 has its own limiting section. As will be explained below, the limiting section of each trap operates substantially as described with reference to Figure 4.

[0108] More specifically, the first channel 101, in particular the input portion 102 of the first channel 101, delivers fluid to each trap. For each trap, the first channel 101 has an input portion, an output (or bypass) portion, and a feeder channel portion. Figure 2 shows the input portion 120a, output or bypass portion 122a and feeder channel portion 116a of the first trap 108a, and the input portion 120b, output or bypass portion 122b and feeder channel portion 116b of the second trap 108b. Multiple traps are arranged in series such that the bypass portion of a trap is coupled to the input portion of a subsequent trap in a series of traps. In this embodiment, the bypass portion 122a of the first trap is coupled to the input portion 120a of the second trap. In the embodiments of Figures 1, 2 and 3, for each trap, the input portion (120a, 20b) is substantially perpendicular to the corresponding bypass portion (122a, 122b). In other embodiments, the input section is positioned at an alternative angle to the bypass section.

[0109] The feeder channel portions (116a, 116b) are sized to receive and hold objects and can function as object containment chambers. Each trap is positioned such that the outlet channel 106 is in fluid communication with the feeder channel portions (116a, 116b) via their respective restricting portions (114a, 144b). Each trap can be considered to have an outlet channel portion that forms part of the outlet channel 106 such that a restricting portion is provided between the feeder channel portion and the outlet channel portion.

[0110] It will be understood that each restriction is formed within a fluid channel defined by the feeder channel portion 116a and a portion of the outlet channel 106. As illustrated with reference to Figure 4, each trap can have a first configuration (referred to as an open configuration) and a second configuration (referred to as a closed configuration).

[0111] In the open configuration, the first channel 101 is in fluid communication with the outlet channel 106 via a restricting section. For the first trap, in the open configuration, a fluid path is defined between the input channel 101 and the outlet channel 106 via a restricting section 114a. Thus, in such a configuration, the fluid can flow from the feeder channel section 116a through the restricting section 116a to the outlet channel 106.

[0112] In a closed configuration, the first channel 101 is not substantially in fluid communication with the outlet channel 106 due to occlusion and / or blockage at the limiting portion 114a. In this embodiment, the occlusion is formed by a tissue spheroid held within the feeder channel portion 116a. The tissue spheroid is either embedded within the chamber or otherwise seeded. In particular, in this embodiment, the tissue spheroid is substantially immobilized within the chamber. It will be understood that the tissue spheroid is immobilized permanently, or at least for a period sufficient to allow a signal to be detected by electrodes. Figure 3 shows an immobilized or captured cell spheroid 120 within the first trap 108a. The cell spheroid described with reference to Figure 3 is merely an example, and it will be understood that in other embodiments, the captured object may be at least one of organoids, tissue spheroids, and / or pancreatic islets, cells, or tissue objects. In some embodiments, the object may be pancreatic islets derived from the pancreas. Since the cell spheroids have dimensions (diameter or width) larger than the opening of the limiting portion 114a, the cell spheroids form a barrier to the fluid flow so as to prevent substantially all of the fluid in the first channel from flowing from the first channel 101 to the outlet channel 106. As illustrated with reference to Figure 4, the traps may be moved from the first configuration to the second configuration by performing a cell spheroid seeding process using the device. If the traps are in a closed configuration, the fluid bypasses the traps and flows into subsequent traps in the row (through the bypass portion of the closed trap and the input portion of the subsequent traps).

[0113] Multiple traps are traps in which one or more of them can be blocked by cell spheroids. If one of the traps is blocked, the fluid flows substantially through one of the unblocked traps through the fluid path defined between the input channel 101 and the outlet channel 106. If all of the traps are blocked, the fluid bypasses all of the traps and flows substantially along the input channel 101 through a further fluid path defined between the input channel portion 102 and the bypass channel portion 104.

[0114] Figure 3 shows a first trap 108a in a closed configuration and a second trap 108b in an open configuration. As shown in Figure 3, the first trap 108a has a cell spheroid 120 in a cell spheroid chamber, which forms a barrier to fluid flow from the cell spheroid chamber side, which is the first side of the limiting section, to the second channel 106 side, which is the second side of the limiting section. In such a closed configuration, the fluid flows along the input channel 106, bypasses the first trap 108a, and then flows into the second trap 108b. More specifically, the fluid bypasses the limiting section, exits the first trap 108a through the bypass portion 122a, and enters the second trap 108b through the input portion 120b. In some embodiments, the fluid flow across the traps in a closed configuration is blocked or at least reduced compared to an open configuration.

[0115] As shown in Figure 3, the second trap 108b, in the open configuration, allows fluid flow from the spheroid chamber side, which is the first side of the restrictor, to the second channel 106 side, which is the second side of the restrictor. In such an open configuration, the fluid flows along the input channel 101, and at least some of the fluid passes through the restrictor 114b to the outlet channel 106.

[0116] In the above embodiment, the restricting portion was described. The restricting portion works in cooperation with the chamber to capture tissue, and can therefore be considered to act together as a capture element configured to capture cell spheroids. In alternative embodiments, it will be understood that alternative restricting portions and / or capture elements may be provided. For example, a mesh or grid structure may be provided to restrict cells and / or other objects or tissue cultures while allowing fluid to flow. Further examples of capture elements are provided with reference to Figure 4.

[0117] During use, multiple objects in a fluid are introduced into the microfluidic device via the input 110. The fluid initially flows through the device along multiple fluid paths via the input section 102 and the limiting section 108. The multiple objects in the fluid then reach the limiting sections, where they are captured and / or at least restricted as described above. Once captured at each limiting section, a detection process is performed to obtain sensor signals of the captured objects using multiple electrodes. In some embodiments, the fluid flow is due to gravity and / or capillary action.

[0118] The detection process may be performed before or after delivering additional fluid to the captured object. The additional fluid is introduced into the device via input 110 and supplied to the captured object via an additional fluid path. The fluid then flows to an additional output 113. The detection process includes detecting sensor signals from electrodes provided in each trap. In this embodiment, the sensor signals represent or indicate the biological activity of a plurality of captured objects, in this embodiment, objects. Before the delivery of additional fluid, a first sensor signal representing or indicating a baseline electrophysiological activity for the plurality of objects may be detected, and further signals representing or indicating the electrophysiological activity of the plurality of objects may be detected in response to the fluid.

[0119] Figure 1 shows a multi-channel microfluidic module, but it will be understood that two or more of these modules can be arranged, for example, as an array, to provide additional channels. Such combinations of modules may also be accompanied by corresponding arrays or correspondingly larger microelectrode arrays, as will be described below.

[0120] Figure 4 (top view) shows a fluid device having a first channel 12 and a second channel 20. Figure 4 is described to illustrate the operation of a single trap for background information. The first channel 12 has a fluid input 14 and a fluid output 16, with a trap provided between the input 14 and the output 16. The trap has a limiting section 18 provided in the first channel 12 between the input 14 and the output 16. The limiting section 18 can be considered to define the feeder channel portion 12a and the output channel portion 12b of the first channel. The feeder channel portion 12a may also be referred to as the feeder channel, and the outlet channel portion 12b as the outlet channel. The limiting section 18 is sized to allow fluid flow between the feeder channel portion 12a and the output channel portion 12b, and to prevent one or more cell spheroids from passing through the limiting section. This limiting section can be considered an opening formed by two members 19a, 19b provided in the first channel. The second channel 20 is in fluid communication with the feeder channel portion 12a and is sometimes referred to as a bypass channel. The trap also has, for example, at least one neutral reference electrode or ground electrode and a measurement electrode (c') for detecting the electrophysiological signals of one or more cell spheroids trapped in the trap. The detection of electrophysiological signals using the trap is described elsewhere.

[0121] It will be understood that the channels define fluid paths through the device. In particular, there is a first fluid path from input 14 to output 16, and the limiter 18 is a limiter on this fluid path. In addition, a further fluid path is defined between input 14 and a second (or bypass) channel 20. The first fluid path uses fluid flow to deliver cell spheroids to the trap limiter. Thus, cell spheroids are prevented from flowing along the first fluid path by the limiter. The further fluid path is in fluid communication with the limiter and can be used to deliver further fluid to the trapped cell spheroids to enable fluid exchange and / or further fluid delivery to the trapped cell spheroids. The first fluid path overlaps with the further fluid path at least partially.

[0122] During use, the trap may be in one of two configurations: an open configuration or a closed configuration. In the open configuration, the feeder channel portion 12a is in fluid communication with the outlet channel portion 12b via the restricting portion 18. In the open configuration, the fluid can flow along the first channel from the feeder channel portion 14 through the restricting portion 18 to the outlet channel portion 16.

[0123] In the closed configuration, the feeder channel portion 12a is not in fluid communication with the outlet channel portion 12b due to occlusion and / or blockage at the limiting section. During use, such occlusion is formed by cell spheroids held in the feeder channel portion 12a at the limiting section 18. Since the cell spheroids have dimensions (diameter or width) larger than the opening of the limiting section, the cell spheroids, together with members 19a, 19b within the channel 12, form a barrier to fluid flow, preventing substantially all of the fluid in the first channel from flowing from the feeder channel portion 12a to the outlet channel portion 12b. As a result, when the limiting section is occluded, the fluid flows into the bypass channel 14 at a faster rate. The trap may be moved from the first configuration to the second configuration by performing a cell spheroid seeding process using the device. It will be understood that in the closed configuration, the fluid flows into the bypass channel at a faster flow rate than in the open configuration.

[0124] As described above, the device is a combination of a transparent microfluidic channel design and a “trap” provided with a printed microelectrode array (Figure 4 shows an example of a single channel). In the embodiment of Figure 4, the channel dimensions of the feeder channel and outlet channel are 450 μm wide and 250 μm deep (Figure 4), and the main channel limiting portion of the trap and bypass channels is 20 μm to 50 μm (a value selected depending on the required flow rate). The width of the limiting portion (i.e., the narrower channel) can be selected from a suitable range of widths to ensure uniform flow in multi-channel systems and single-channel systems. In some embodiments, the width range includes 20 μm to 50 μm. Waste, which can also be sampled to evaluate the secretion of insulin or other substances, exits the system through the output channel (Figure 5).

[0125] Figure 4 provides additional details about the arrangement and dimensions of the channel microfluidic and electrode array (showing a single electrode as part of a single channel example). Specifically, a: input channel dimensions 450 μm, b: limiting section width 40 μm, c: reference electrode, c': measurement electrode, d: profile in cross-section, e: PDMS channel cross-section, f: silicon oxide insulation (thickness 100 nm (not to scale)), g: gold electrode track and electrode pad, h: glass substrate, I: channel height (250 μm), j: channel width (450 μm as shown in a).

[0126] Figure 5 provides a simplified example of further details of the channel arrangement, where a: the arrangement of the input and output channels of a single microfluidic trap shown in Figure 4, with bypass arrows indicating the direction of flow; b: a diagram of the channel bypass of a multi-channel microfluidic device, with arrows indicating the direction of flow (the channel dimensions are the same as those of a single-channel device); c: a photograph of the assembled multi-channel electronic chip with the microfluidic module mounted; d: the multi-channel microfluidic chip and electrode array mounted on the holding module; and e: further photographs of the microfluidic chip and electrode array.

[0127] Figure 6 shows a detailed diagram of the glass substrate on which the electrode array is printed, and demonstrates an example of the functionality of the multichannel device, with a: an overview of the alignment of the diagram showing the electrodes and microfluidic circuit, b: details of the electrodes and microfluidic traps, e': electrode track, f': reference electrode, g': microfluidic channel, c: a channel like the one in a, showing the capture of a 150 μm glass sphere (h'), d: linear channel arrangement showing microfluidic capture of a 150 μm glass sphere on the electrode, and e: a composite micrograph showing an example of a microfluidic module after assembly and pancreatic islets captured on a microelectrode array.

[0128] In some embodiments, one or more microfluidic channels deliver pancreatic islets to the “trap” using capillary action without the need to apply negative or positive pressure (see, e.g., Figures 4, 5, and 6e). Once the islets are trapped, the main channel is substantially blocked, and a microfluidic bypass allows the test solution to pass through (see, e.g., Figures 4 and 6e).

[0129] Such permeability allows for rapid exchange (again, by capillary action and / or gravity) between normal extracellular solution and a solution containing a higher concentration of glucose (e.g., 15.5 mM glucose), washing for aggregation or evaluation of objects, and labeling with drugs or dyes. As glucose concentration increases, glucose detection induces an increase in the electrical activity of pancreatic beta cells in the object (Figures 10a and 10b). Since beta cells constitute the majority of glucose-sensing cells in the islets, the measurable electrical change in activity can be detected by a 30 μm electrode pad on which the islets are immobilized, relative to a larger "ground" electrode (Figure 4). This electrical response is observed only in healthy, fully functional islets. The electrical output of the chip is "read" by connecting the array via electrical contact with a large printed metal contact pad connected to an electrode pad located beneath each islet via an insulating path. Processing of the electrical signal provides an indicator of whether or not the islets are responding.

[0130] The device is proposed to enable timely assessment of the viability of islet batches. A prototype has been constructed and used to assess islet quality (Figure 9D, Figure 10a, and Figure 10b). One batch of islets is expected to take approximately 30 minutes to assess challenges related to low glucose, followed by high glucose. These results will then be compared with other viability assays.

[0131] It is assumed that islet samples from the donor pancreas can be pipetted into such a device and separated and immobilized on an electrode using a microfluidic channel and trap. Glucose addition and monitoring of electrode activity will be used to obtain % viability using root mean squared noise (RMS) measurements. This process will take up to 2-3 hours. Currently, in human islet experiments, islets are cultured for up to 48 hours, so the timeline should fit well within this margin.

[0132] The device will be understood to be a hybrid electronic and microfluidic device. Figure 5(c) is an image of the device in use according to one embodiment. The device has a microfluidic module 52 and an electronic module 54. Means for supplying fluid to the microfluidic module, a pipette 56 in this embodiment, are also shown. The microfluidic module has a number of limiting sections, as substantially described above with reference to Figures 1 to 3, for example.

[0133] Figure 5(c) is a first photograph of the device including the electronic module 52 and the microfluidic module 54, and Figure 5(e) is a second photograph of the device including the electronic module 52 and the microfluidic module 54. The microfluidic module 52 may optionally be referred to as a microfluidic chip together with the electronic module. Figure 5(d) also shows a holder 56. The holder, also called a chip holder, is sized and shaped to provide a base for the chip. In particular, the holder is substantially U-shaped. The gap allows the chip to be inspected when held within the holder. The gap has a rim to provide a base for the device, in particular the microelectronic module. When the microelectronic module is held by the holder, it will be positioned on the rim of the holder, and the microfluidic module will be positioned on the microelectronic module.

[0134] Figure 5(e) additionally shows input port 58, first output port 60, and second output port 62, corresponding to input port 1308, first output port 1302, and second output port 1304 described with reference to Figure 13. In this embodiment, input port 60 (also called the inlet) has a diameter of 1 mm, and output port 62 (also called the flushing outlet) has a diameter of 1 mm. The second output port 64, also called the waste outlet, has a diameter of 6.0 mm. The inlet and outlet are formed by providing voids or notches in the PDMS layer.

[0135] Figures 7(a) and 7(b) show schematic diagrams of the electrode configuration of the electronic module. This is a glass electrode array for a 15-channel device according to one embodiment. The sensing module is provided on a glass substrate layer 702 with dimensions of 5 × 5 cm. The thickness of the glass substrate layer is 1.5 mm. The electrode configuration 704 is patterned on the glass substrate layer. Figure 7(b) shows an enlarged view of the electrode configuration 704. Multiple electrodes (labeled 1 to 15 in this embodiment) are provided so that their respective endpoints are in a linear arrangement and define a straight line. The end of the first electrode 708 is shown in Figure 7(b). A single reference electrode 706 spans the width of the multiple electrodes. The electrode configuration is operable to detect a signal for each electrode by reference to the reference electrode. Each electrode, when used with the microfluidic module, is aligned at or adjacent to its respective limit so that a signal for a captured object can be detected. Figure 6(e) above shows a captured pancreatic islet with electrodes.

[0136] It will be understood that the device may have a different number of channels and limiting elements. Figure 8(a) shows an electrode configuration for a 60-channel device. Figure 8(a) shows an electrode configuration with four reference electrodes. Each reference electrode is grouped with a set of 15 sensing electrodes, as described with reference to Figures 7(c) and 7(d). In the embodiment of Figure 8(a), it will be understood that four sets of 15-channel microfluidic modules are combined for use with the electrode configuration. The sensing module in Figure 8 is mounted on a 10 cm × 5 cm glass substrate. Figure 8(b) shows two sets of 15-electrode modules together to form part of a 30-channel device. In both Figures 8(a) and 8(b), the modules are arranged so that the reference electrodes within each module are parallel. In both Figures 8(a) and 8(b), the modules are arranged so that the linear arrangement of the electrode ends is parallel.

[0137] It will be understood that sensor signals can be detected between a reference electrode and each trap electrode. In some embodiments, the sensor signal for each trap is detected during the detection process. In some embodiments, the signals from each trap / channel are isolated from each other. The reference electrode may be a ground reference electrode. The positioning of the ground electrode may be done such that the ground reference electrode overlaps with at least a portion of further fluid paths and / or multiple fluid paths.

[0138] In the embodiments described above, hybrid microfluidic and electronic devices have been described. At least a portion of the device may be provided as a cartridge for use with further devices. Figure 9 shows a device 900 according to one embodiment. In Figure 9, the processing resources are provided as part of a larger device configured to accept a removable chip. The removable cartridge or chip may correspond to a device, as described above. In some embodiments, it is a removable cartridge.

[0139] The device 900 includes one or more displays 902, a tip holder 904 having a housing 904, and a processing resource provided as part of a data processing PC 908 in this embodiment. The device is configured to receive disposable tips 906 through a first cartridge opening 908 on a first surface which is the top surface of the housing 904. One or more fluid openings may be provided on the top surface of the housing. In this embodiment, the fluid openings are sized to receive a pipette. In this embodiment, the microfluidic module of the cartridge includes 60 channels (in four separate groups, each group having a separate fluid inlet). A manifold is therefore provided as part of the device or as a removable part used with a pipette, to guide the received fluid to each of the separate fluid inlets. In some embodiments, the fluid inlets and cartridge opening are aligned so that, when inserted into the cartridge opening, the inlets of the microfluidic module are aligned with the fluid opening, enabling the delivery of fluid to the inserted cartridge. The processing resource may be configured to run software for automated or at least partially automated data analysis of the sensing signals from the cartridge.

[0140] Further devices may also include a user input device (not shown) for enabling a user to interact with and control the device. The user input device may enable the user to control the sensing process and / or control the data analysis and / or select various results to be displayed. In a non-limiting example, in some embodiments, the device may be operable to enable the user to select the number of channels to be used (e.g., up to 60) via the user input device.

[0141] The apparatus may further include a camera or other suitable imaging means for enabling image acquisition of the cartridge during use. The apparatus may be operable, for example, to allow selection of another acquired image during data acquisition. This selection may be made using a user input device. The apparatus may further be operable to allow the user to perform the test on a subset of available traps. For example, the user may determine that islets are present only in a subset of traps and thereby choose to perform the test only on those channels.

[0142] The display may also be configured to display results. In this embodiment, the display displays the percentage of viable pancreatic islets. In some embodiments, the device may be operable so that the user can select the results to be displayed using a user input device. The user input device may be any suitable user input device such as a mouse or keyboard. In some embodiments, the display forms part of the user input device.

[0143] In some embodiments, at least a portion of the cartridge is disposable. For example, microfluidic components may be disposable.

[0144] In some embodiments, the removable cartridge contains only the microfluidic portion of the device, and the electronic module is provided separately (for example, as part of the device or as a further component to be combined with the cartridge).

[0145] Although not shown in Figure 9, there are additional computing and / or storage resources provided within the housing. For example, in some embodiments, it will be understood that the device may have memory storage for storing data and / or be connected to a network to enable the transfer of data from the device to further computing devices. In some embodiments, the device may have removable storage media. Amplifiers may be provided to amplify one or more signals detected by electrodes.

[0146] The processing resource is configured to process signals from electrodes or data derived from those signals. Processing of signals and / or data may be for determining the percentage of viable objects in the sample, such as tissues and / or cells. Alternatively, or in addition, processing of data may be for determining at least one of electrical activity, viability, function, and / or response to stimulation, and may include generating and storing data representing electrical activity, function, and / or response to stimulation.

[0147] In some embodiments, the processor is configured to determine a measure of the electrical activity of a captured object in response to further fluid introduced through a further fluid pathway. The processor may also be configured to determine the number of viable and / or functional tissues and / or multicellular objects in a sample, or to make a diagnosis and / or indication of viability based on the detected electrical activity in a plurality of limiting and / or sensing regions, in which case, for example, the diagnosis and / or indication of viability may be based on the electrical activity in response to further substances, optionally including chemicals and / or drugs, such as glucose.

[0148] While glucose is described as an example, it will be understood that other types of further fluids are available. In particular, a washing solution may be introduced to remove any solution from the device. Alternatively, molecules may be introduced to label the captured object, or one or more drugs and / or cell activity stimulants or inhibitors may be introduced.

[0149] Figure 10 shows the results of electrical measurements from human pancreatic islets. a: Pancreatic islets were exposed to 3 mM glucose, followed by 15 mM glucose. The glucose-stimulated insulin secretion (GSIS) measurement shows the root mean squared (RMS) of the electrical response in the upper trace at 15 mM glucose and the electrical noise in the lower trace at 15 mM glucose. Note that there is no insulin secretion at 3 mM glucose, and therefore no corresponding bioelectrical activity. Furthermore, b: shows the results of GSIS stimulation with 5.5 mM to 15.5 mM glucose, c: shows the results of RMS measurements to show the sensitivity of the GSIS response with 7.3 mM glucose and then 17.3 mM glucose (the results showed a significant difference between the two glucose concentrations in Student's paired t-test (N=11) at a P<0.0001 level), and d: shows the viability (%) of the tested islets. Here, three batches of islets were examined in response to 3 mM glucose and then 15 mM glucose, and the number of islets per batch that showed electrical activity in response to 15 mM glucose was calculated and expressed as a percentage for each of the three batches of islets (N islets tested per batch = 16, 13, and 16, respectively). Note that the 3 mM glucose solution acts as a control condition in which insulin secretion or electrical activity of the islets is not expected.

[0150] Figure 11(a) shows a device containing a microfluidic module along with a sensing element module, as described with reference to Figures 1-3. Figure 11(c) shows the device from Figure 2 with a captured object, in this case a spheroid. Figure 12 shows a cross-section of the device along the line XX′ marked in Figure 11(a). The device in Figure 11 has the same features as those described with reference to Figure 1, although some reference numerals have been omitted for clarity.

[0151] Figure 11(a) shows the first electrode 1102, the second electrode 1104, and the reference electrode 1106. It will be seen that the first electrode 1102 is aligned with the first trap 108a, and the second electrode 1104 is aligned with the second trap 108b. In particular, the first electrode 1102 is aligned along the opening of the first limiting portion of the first trap 108a, and the second electrode is aligned along the opening of the second limiting portion of the second trap 108b.

[0152] The first electrode has a first elongated portion 1102a and an electrode end portion 1102b. It will be understood that at least a portion of the elongated portion corresponds to an electrode track or trace, and only a portion of the elongated portion 1102a is shown in Figure 11(a). The elongated portion is parallel to the opening of the limiting portion, and in particular, when viewed from above, the elongated portion appears to pass through the opening of the limiting portion. Thus, when the limiting opening is centered on the limiting axis and the elongated portion is aligned with the electrode axis, the limiting axis and the electrode axis are parallel. It will be understood that the second electrode is aligned with the corresponding second limiting portion of the second trap. However, it will be understood that one or more of the electrodes, for example, the first electrode and the 15th electrode in Figure 7(b), do not need to be aligned with the limiting portion. In some embodiments, the elongated portion may be an electrode track, and the end portion may be referred to as an electrode pad. In some embodiments, the elongated portion corresponds to an electrode trace, and the electrode end portion is the electrode itself or includes at least the sensing portion of the electrode. As explained with reference to Figure 12, the elongated portion is insulated by an insulating layer, and it will be understood that the electrode ends come into contact with the fluid and / or the captured object itself during use.

[0153] The electrodes are arranged such that each electrode end portion is substantially located in the area corresponding to the trap chamber. The ground electrode 1106 is substantially located along the delivery portion of the further fluid path and along multiple fluid paths. Thus, a distance is provided between each electrode end and the reference electrode. Figure 11(b) shows the device of Figure 11(a) with two captured objects 1110a, 1110b in the capture position.

[0154] Figure 12 shows a cross-sectional view of the electrode arrangement within the trap at cross-section XX′ of Figure 11a. As seen in Figure 12, the device has a PDMS layer 1202, an insulating layer 1204 (a silicon oxide insulating layer in this embodiment), and a glass substrate layer 1206. In this embodiment, the insulating layer 1204 is located between the PDMS layer and the glass substrate layer 1206. As described above, several channels are formed in the PDMS layer to form a microfluidic module. Figure 12 also shows the chamber 1208 of the trap defined within the PDMS layer by the channels. The chamber has a height and width. The channels forming the chamber are 250 μm high and 450 μm wide.

[0155] Figure 11b also shows the position of the electrode relative to the trap and the captured organoid 1110b. The electrode is placed on a glass substrate, and a portion of the electrode is insulated by an insulating layer. Figure 12 shows the first electrode 1102 on the glass substrate layer 1206, having an elongated portion 1102a and an electrode end 1102b, as described with reference to Figure 11(a). The electrode is positioned below the chamber such that the electrode end is located in the central portion. In this embodiment, the electrode end, which is the non-insulated portion of the electrode, is exposed to the upper channel. During use, in this embodiment, the non-insulated portion of the electrode is in contact with the captured object in the trap. In some embodiments, the non-insulated portion of the electrode is adjacent to the captured object and is in contact with the fluid containing the captured object.

[0156] It will be understood that a microchannel module, for example, the module described with reference to Figure 1, is formed in a PDMS layer. Figure 13 shows a module according to one embodiment. In addition to the features described with reference to Figure 1, the module of Figure 13 also has an inlet port 1308, a first output port 1302, and a second output port 1304. The inlet port is an inlet provided at the fluid input, as described with reference to Figure 1. The first output port is an outlet provided at the shared output, as described with reference to Figure 1, and may also be referred to as the waste output. The second output port is an outlet provided at the first output, as described with reference to Figure 1, and may also be referred to as the flushing output. Each of the inlet port and the outlet port is circular in this embodiment. The channels and ports of Figure 13 are formed in a PDMS layer 1310 having a width and length of 3 cm and a height of 0.5 cm.

[0157] Figures 14(a) and 14(b) show an electrode array and associated microfluidic module according to a further embodiment. The device in Figure 14 has an additional stimulating electrode, which, when combined with a ground electrode to form a circuit, is operable to deliver pulses of electrical stimulation across the captured object.

[0158] As can be seen from Figure 14(a), the electrode array is substantially the same as described with reference to Figure 7, with the following differences. As described with reference to Figure 7, the multiple electrodes 1702 are arranged such that their respective endpoints are in a linear arrangement. These endpoints may form part of their sensing element. In this embodiment, the central electrode (labeled 1404) of the multiple electrodes is the reference electrode. The reference electrode is the ground electrode. In this embodiment, the single electrode 1406 spans the width of the multiple electrodes and is the stimulating electrode. As can be seen from Figure 14, the central electrode 1404 is wider than the other electrodes of the multiple electrodes. In the embodiment of Figure 14, the reference electrode and the stimulating electrode form a stimulating electrode pair, and the sensing electrode is configured to detect a response from the stimulating signal generated by the stimulating electrode pair.

[0159] As described with reference to the embodiments described above, the electrode array is aligned with the microfluidic device 1408, which is substantially as described with reference to Figure 1, for example, with the following differences. Similar to the microfluidic device described above, the second channel of the microfluidic device of Figure 14 has a first portion 1416a and a second portion 1416b. In this embodiment, the second channel 106 remains coupled to the first channel along the first portion 1416a via a plurality of restrictors, but the second portion 1416b is located in the center of the first portion. The bypass portion and delivery portion remain in the same configuration. The central reference electrode is aligned so that its length is parallel to the second portion 1416b located in the center of the microfluidic device. In this embodiment, a plurality of fluid paths are defined between an input (1422) and an output (1424) via restrictors, and the fluid paths comprise portions of the first portion 1416a and the second portion 1416b. The limiting section is aligned along the axis, and the stimulating electrode is positioned parallel to that axis. The stimulating electrode overlaps with multiple fluid pathways.

[0160] The electrode configuration is operable to detect signals for each electrode by reference to a reference electrode. In contrast to the embodiments described above in which each electrode is aligned at or adjacent to its respective limit to enable passive detection of signals for a captured object, the embodiment in Figure 14, specifically a single electrode 1406, is configured to generate a stimulating electrical signal. Thus, the single electrode 1406 may also be referred to as the stimulating electrode. The multiple electrodes 1402 may also be referred to as sensing electrodes or recording electrodes.

[0161] Electrical stimulation can directly induce simultaneous action potential discharges within an object that can be measured and processed by recording electrodes. For example, direct stimulation can be used in "pacing" myocardial spheroids and other excitable tissues.

[0162] By aligning the electrode array with the limiting section of the microfluidic device, it becomes possible to deliver a high current density to the object through this limiting section.

[0163] Figure 14(b) shows a magnified view of the array and microfluidic device of Figure 14(a). It can be seen that the electrodes are arranged such that there is a distance between the stimulating electrode 1402 and each of the multiple sensing electrodes in each chamber / sensing region / limiting area. In this embodiment, the distance 1410 is shown between the endpoint of the sensing electrode (e.g., 1402i) and the nearest portion of the stimulating electrode 1406. Due to the electrode arrangement, this distance is the same for each stimulating electrode. In use, for each chamber, the stimulating electrode stimulates electrical activity across this distance within the chamber, and the corresponding sensing electrode senses the electrical response across this distance due to the contents of the chamber. The electrical response may be a measured voltage. A central reference electrode measures a reference voltage that can be used as a reference for each measurement.

[0164] In this embodiment, a single stimulating electrode positioned perpendicular to multiple sensing elements is described. In other embodiments, different electrode arrangements may be used. For example, multiple stimulating electrodes may be provided, and / or pairs of stimulating electrodes and sensing electrodes may be provided.

[0165] A non-limiting method using the device in Figure 14 is described below. First, one or more objects are captured as described above. The captured objects in each chamber may be positioned on the passive electrode (or in the sensing region of the chamber around the passive region). The passive electrode is configured, for example, to detect electrical activity in the sensing region after a time delay. An electrical stimulus is delivered to the chamber via a pair of stimulating electrodes that elicit a response in the object in this embodiment. The response is measured by the passive electrode. It will be understood that by adding a pair of stimulating electrodes (in this embodiment, a reference electrode and a stimulating electrode), the device can be used to test several excitable cells / objects and experimental scenarios, such as drug testing and screening.

[0166] The following non-limiting examples and experimental results are described below. I. Qualitative Islet Response Visual evaluation was used to identify responses and no responses to glucose challenges from each protocol used across the three tested islet batches. Differences in glucose-inducible electrical burst activity and inactivity periods between protocols (3 mM–15 mM, 5.5 mM–15.5 mM, and 7.3 mM–17.3 mM) were also visually evaluated. For all protocols used across the three batches (3 mM–15 mM, 5.5 mM–15.5 mM, and 7.3 mM–17.3 mM), the inventors identified either an electrical response or no response by the islets to increasing glucose concentration. In electrophysiological experiments with the 3 mM–15 mM protocols, the islets were initially inactive and then responded to glucose addition (10a), or showed a baseline response to 5.5 mM glucose and a high response to 15 mM glucose (10b). No response was observed in the continuous islet electrical activity following tolbutamide treatment from any of the glucose increasing protocols. Gaps in recordings at different time points reflect artifacts from glucose addition that were "blanked" due to different recording lengths.

[0167] II. Pancreatic Islet Response Pancreatic islets were initially analyzed for electrical activity, and then evaluated for the increase in electrical activity after glucose addition. Electrical response (μV) was quantified using the RMS output (described above) by evaluating samples at 2 minutes (for 10-minute recordings), 3 minutes (for 20-minute recordings), or 5 minutes (for 30-minute recordings) before and after glucose addition for all islet batches (1, 2, and 3). Only data from islets confirmed to be present on the electrodes were included in the dataset.

[0168] III. Background Electrical Activity Before quantifying the electrical response of pancreatic islets, they were first characterized as either electrically active or inactive. If electrical activity was present, the islet was considered active. Where inactive MEA channels were present, recorded activity was compared to inactivity. No intermediate category of dying but active (indicated by continuous activity unrelated to glucose (non-glucose responders)) was observed. All islets (n=16) tested in Batch 1 using the 7.3mM–17.3mM glucose protocol showed electrical activity (100%). In Batch 2, all islets tested using the 7.3mM–17.3mM (n=8) and 3mM–15mM (n=16) glucose protocols showed electrical activity (100%). All islets tested using the 5.5mM–15.5mM protocol from Batch 3 (n=24) showed electrical activity (100%). These results indicate that some electrical activity can be detected in pancreatic islets even at low glucose or control glucose levels.

[0169] IV. Glucose-Induced Electrical Activity After verifying the ability of RMS function to reflect changes in electrical activity associated with high glucose concentrations, this was used to determine whether electrically active pancreatic islets responded to glucose and whether they were viable or unresponsive. Of the 16 islets tested using the 7.3 mM–17.3 mM protocol in Batch 1, 9 showed an increase in RMS. In Batch 2, of the islets tested using the 7.3 mM–17.3 mM protocol (n=8) and the 3 mM–15 mM protocol (n=16), 2 and 11 arelets, respectively, showed an increase in RMS after the glucose challenge. In Batch 3, when using the 5.5 mM–15.5 mM protocol, 16 out of 24 islets tested showed an increase in RMS. As a result, the total percentages of islets showing an increase in RMS value for the 3mM–15mM, 7.3mM–17.3mM, and 5.5mM–15.5mM protocols were 68.75%, 45.83%, and 66.66%, respectively (Figure 7d). From Batch 2, there was one islet tested using the 3mM–15mM protocol, which showed a slight increase in RMS value of 3%, but was not classified as responsive to glucose. Islets classified as unresponsive to glucose showed no change or a decrease in RMS value when the glucose concentration was increased. Next, it was determined whether the increase in RMS value when challenging the islets with high glucose concentrations was statistically significant. Only RMS values ​​of islets showing an increase were included. When present in the culture medium, the mean RMS values ​​(n=11) of pancreatic islets tested using the 7.3 mM–17.3 mM protocol showed a significant increase (paired t-test, t=4.625, df=10, p=0.0009) between glucose concentrations of 7.3 mM and 17.3 mM. The mean (±SEM) RMS of pancreatic islets at a glucose concentration of 7.3 mM was 3.95±0.72 μV, and this significantly increased to 10.13±1.82 μV when the glucose concentration increased to 17.3 mM. The RMS values ​​of the electrical response ranged from 8.87 μV to 21.9 μV for 7.3 mM glucose and 17.3 mM glucose, respectively.Therefore, the majority of the pancreatic islets tested responded with a measurable electrical response to the increase in glucose, which was detectable by measuring RMS noise.

[0170] V. Survival rate of pancreatic islet batches The survival percentage for each batch was calculated by identifying islets that were electrically active and showed an increase in their RMS value after glucose challenge using one of the following glucose protocols: 3 mM–15 mM, 5.5 mM–15.5 mM, and 7.3 mM–17.3 mM. Islets were characterized as viable because the increase in RMS value of islets in response to glucose was significant in the 7.3 mM–17.3 mM, 3 mM–15 mM, and 5.5 mM–15.5 mM protocols. As a result, the batch survival percentages were calculated as 56.25% for batch 1 (9 out of 16 islets tested), 54.16% for batch 2 (13 out of 24 islets tested), and 66.66% for batch 3 (16 out of 24 islets tested). There were differences in the survival percentage depending on the day of testing. In batch 1, 15 islets were tested after 3 days of culture, and 9 (60%) were viable (indicating an increase in RMS). One islet tested after 4 days of culture was not viable (indicating a decrease in RMS). In batch 2, 15 islets were tested after 1 day of culture, and 4 (26.66%) were viable. All 6 islets tested after 2 days of culture were viable (100%). An additional 3 islets were tested after 6 days of culture, and all 3 were viable (100%). From batch 3 onwards, 5 islets were tested after 1 day of culture, and 3 were viable (60%). This increased to 80% viability (4 out of 5 islets) after an additional 2 days of culture. After 5 days of culture, this decreased to 61.5% (8 out of 13 islets). Therefore, rapid measurement of electrical activity using a composite chip allows for differentiation between islet batches and islet viability.

[0171] This disclosure demonstrates that the electrical activity of islets differs from batch to batch per islet isolate, and that prolonged oxygen deprivation impairs islet function, resulting in a lack of response to glucose challenge, i.e., reflecting unviable islets. These two observations indicate that electrical activity in response to glucose challenge is a good surrogate indicator for rapidly determining the health and functionality of islet batches. This novel device successfully captures islets in a way that facilitates simultaneous recording and querying of multiple islets via multiple channels. This is envisioned to form the core technology for a new benchtop device for determining islet quality before transplantation. Such a device could be used in islet transplantation laboratories worldwide. This would also lead to the standardization of islet evaluation and enable comparisons between different laboratories. Means to improve isolation and handling techniques will ultimately lead to efficient techniques for selecting functional islets from non-functional ones. Development of pancreatic islets derived from human embryonic stem cells is progressing, and early Phase 1 clinical trials are underway in humans. Therefore, there is an objective need to evaluate the viability of such stem cells, and there is ample room for the use of such devices in this field as well. Furthermore, such devices may be used for clusters of cells derived from other tissues to test their viability.

[0172] The following comments are provided regarding the details of the materials and manufacturing methods according to the embodiment. Manufacturing method and usage method I. Design of Microelectrode Arrays (MEAs) The custom MEA was designed using AutoCAD (Autodesk, Inc., California, USA). Several different electrode configurations proved suitable (e.g., Figure 6A). The design in Figure 6A features a total of 11 recording electrodes and an integrated ground reference electrode (details in Figure 6b). The recording electrodes had a diameter of 30 μm, and the distance between the recording electrodes and the ground electrode was 50 μm. The AutoCAD design was sent to Micro Lithography Services Limited (Chelmsford, UK) to produce a film or glass photomask.

[0173] II. Manufacturing of microelectrode arrays (MEAs) The MEA was fabricated at the nanofabrication facility of Heriot-Watt University. Borosilicate was used as the substrate (h' in Figure 6). The borosilicate was cut into 49 × 49 mm sections using a DAD3220 wafer dicing saw (DISCO Corporation, Tokyo, Japan). The electrode design and insulation pattern were transferred onto the borosilicate using photolithography. The electrodes and electrode tracks were deposited with Ti (g in Figure 6), while the insulating portion of the tracks, excluding the electrode tips and pads, was deposited with SiO2 (f in Figure 6).

[0174] III. Spin coating of photoresists Prior to deposition, the borosilicate substrate was cleaned with acetone and diluted Decon 90 (Decon Laboratories Ltd (East Sussex, UK)). It was then rinsed with deionized (DI) water and blow-dried using a filtered air gun. This was done to ensure the borosilicate surface was completely clean and dust-free before starting the process. The borosilicate blank was then placed on a spin coater SPIN150 (SPS Europe (Putten, Netherlands)) and held in place by a vacuum seal. The negative-type photoresist AZ nLOF 2070 (MicroChemicals (Ulm, Germany)) was carefully applied to the entire surface of the borosilicate, taking care to avoid air bubbles. It was then spun at 3500 rpm for 40 seconds. The negative-type photoresist, which would normally form a 7 μm thick layer, was pre-diluted to form a 1 μm layer after spinning. After this process, the substrate was hard-baked at 95°C for 3 minutes.

[0175] IV. Alignment and UV exposure The next step involved aligning one of the multi-electrode film photomasks with the prepared substrate. This was done using an MJB3 Mask Aligner (S.SS MicroTec (Garching, Germany)). The film photomask was placed on the mask holder and then loaded and secured onto the aligner. The substrate was positioned on the substrate holder that had held it during placement using vacuum suction, and then slid under the photomask. The substrate was brought into contact with the film photomask, and then the separation lever was operated to allow alignment of the design with the substrate. Alignment was performed by adjusting the x and y axes in micrometer increments. Once alignment was achieved, the separation lever was pushed back so that the substrate was in contact with the film photomask again. The substrate was then UV exposed for 40 seconds, and upon completion, the substrate was carefully removed from the mask aligner. Post-exposure baking was then performed at 115°C for 90 seconds.

[0176] V. developing Next, the substrates were developed using AZ726MIF (metal ion-free) developer (Microchemicals). Initially, they were left in the developer for 60 seconds, and then carefully monitored for another 15-30 seconds until the design features became visible. The substrates were washed with diluted Decon90 for approximately 30 seconds, then rinsed with deionized double distilled water, and blow-dried with a filtered air gun.

[0177] VI. Electrode deposition After the substrate was deposited, Ti was deposited using a technique called physical vapor deposition (PVD) with a Minilab 080 (Moorfield Nanotechnology, Knutsford, UK). This involves directing an electron beam onto a metal and then depositing the metal onto the substrate. The borosilicate substrate was placed in the outer chamber with the side to be coated facing downwards. Before sliding the substrate into the main chamber containing the Ti-filled crucible, the outer chamber had to be degassed to reach the same vacuum level as the main chamber. During this operation, the necessary parameters were entered into an SQM-160 rate / thickness film deposition monitor (Inficon, Bad Ragaz, Switzerland). These included the thickness of the metal layer to be deposited, the density of the metal to be deposited, and the Z ratio (Z ratio). The thickness was set to 10k (1000nm), the Ti density to 4.500, and the Ti Z ratio to 0.628. Once the substrate was secured and the main chamber door was locked, the electron beam was directed onto the Ti using the controller knob. By slowly increasing the power to a maximum of 100 mA before opening the shutter, the Ti could be deposited onto the borosilicate substrate. The rate was monitored and maintained between 2 and 3 mA / s. When the Ti layer reached 1000 nm, the shutter automatically closed and the electron beam was turned off. The substrate was then carefully removed from the main chamber and moved to the outer chamber, which was then pressurized to room temperature before being opened to retrieve the substrate. Due to electrode deposition, a layer of Ti was formed across the entire surface of the borosilicate. Therefore, the next step involved removing the excess Ti so that only the array design remained. This was done by dissolving the pre-coated negative photoresist using TechniStrip NI555 (MicroChemicals) at 80°C until all the unwanted Ti layers were removed. Dissolving the negative resist also meant that the Ti layer deposited on top of it was removed, leaving only the Ti array design.

[0178] VII. Second spin coating The next step was to prepare the surface for the deposition of the silicon dioxide (SiO2) insulating layer. The photolithography process was similar to that described in the paragraph above. However, this time, an insulating design photomask was used instead, and AZ1505 positive photoresist (MicroChemicals) was used. The spin coater was set to 2000 rpm for 30 seconds. The substrate was also developed in AZ351B Developer MIC (containing metal ions (MicroChemicals)) used in a 1:4 dilution (1 part developer and 4 parts DI water). The substrate was then washed as described above and removed for SiO2 deposition.

[0179] VIII. Insulator deposition SiO2 was deposited using the same process as for Ti, but the density and Z ratio parameters were changed to 2.648 and 1.000, respectively. The thickness of the SiO2 was 500 nm. Since the electrodes and contact pads were areas that needed to be exposed, the next step after deposition was to remove the SiO2 from the electrodes and contact pads. In this case, the positive photoresist was removed using acetone, leaving only SiO2 on the track connecting the electrodes to the contact pads.

[0180] IX. Alternative laser lithography After testing these custom MEAs, it was found that the tracks were not always completely insulated, or the electrodes were not exposed. These problems were solved by using the DWL66+ (Heidelberg Instruments Mikrotechnik, Heidelberg, Germany) 2.5D laser lithography system. This machine can laser etch structures down to 300 nm and can perform direct photoresist patterning. This meant that the step of aligning a film photomask and exposing it to UV was no longer necessary. The array design was tuned using KLayout (Matthias Koefferlein, Germany) for reading by laser writer software. A borosilicate substrate pre-spin-coated with a layer of negative photoresist was placed on the stage, and the rest was done by the software package provided by Heidelberg Instruments. The writing head automatically aligned itself with the substrate before starting the etching of the array design onto the photoresist. Once this was complete, the same steps as previously described for Ti deposition were performed, but this time the Ti thickness was set to 150 nm. Instead of using a film photomask, the insulating design was also laser-etched. This means that the electrodes and contact pads were precisely cut out and therefore exposed after the deposition (100 nm) and removal of SiO2.

[0181] Design and manufacturing of microfluidic microchannels I. Fabrication of microchannels using soft lithography The microchannels were designed using Adobe Illustrator (Autodesk) and then sent to Micro Lithography Services for fabrication as film photomasks. The features of this design are shown in Figures 1, 2, 3, 4, and 5. The soft lithography protocol (MicroChem (2015)) used to prepare the PDMS device was similar to the photolithography protocol used to fabricate the custom MEA, but with different parameters. Instead of a borosilicate substrate, a test-grade silicon wafer (SILI-0005 (PI-KEM)) with a diameter of 7.62 cm and a thickness of 380 μm ± 50 μm was used.

[0182] II. Spin Coating and UV Exposure A silicon wafer was placed on a spin coater (WS-650MZ-32NPP (Laurell Technologies Corporation)) and held in place by vacuum. Negative photoresist SU-8 2025 (Microchem) was spun on the wafer in two stages. In the first stage, it was spun at 500 rpm for 10 seconds at an acceleration of 100 rpm / s, and in the second stage, it was spun at 3000 rpm for 39 seconds at an acceleration of 300 rpm / s. These settings resulted in a film thickness of 100 μm. This process was repeated three more times to obtain thicknesses of approximately 250 μm to 300 μm. The wafer was soft-baked at 65°C for 1 minute, then at 95°C for 5 minutes, and then returned to 65°C for another soft-bake for 1 minute. Next, the designed film photomask was carefully placed on the wafer, ensuring that it did not move once in contact. Then, the wafer was exposed to UV for 41 seconds, followed by a post-exposure bake at the same temperature and time as the soft bake. The transparent parts of the photomask (two sections and microchannels) were crosslinked, while the dark parts of the photomask were not.

[0183] III. Development The wafer was immersed in SU-8 developer and sonicated for 60 seconds. Then it was washed with isopropanol and blow-dried with an air gun. The wafer was returned to the developer for another 60 seconds, but without sonication. The same washing steps were repeated until the wafer was clean. If streaks were still visible, the wafer was returned to the developer for another 60 seconds, and this process was repeated until the streaks disappeared. Finally, the wafer could be used as a mold for manufacturing microchannels, as long as it could be kept intact. The wafer surface is hydrophilic, which means that it would be difficult to peel the PDMS from it without leaving residue or damaging the wafer. A drop of trichloro(1H,1H,2H,2H-perfluorooctyl)silane (448931, Sigma-Aldrich (Missouri, USA)) was added to the wafer, the lid of the petri dish was securely closed, and it was left to evaporate at room temperature in a fume hood. The purpose of this was to make the surface hydrophobic, thereby avoiding the aforementioned problems. The wafer was held in a glass Petri dish and bonded to it with Araldite.

[0184] IV. Preparation of PDMS PDMS was prepared by mixing Sylgard184 silicone elastomer base with a curing agent in a 10:1 ratio. The PDMS was then poured onto the wafer and placed in a vacuum chamber to remove air bubbles before curing. The PDMS was cured at 60°C for 1 hour. After curing, the PDMS module (microchannel) was carefully cut out using a scalpel, taking care not to damage the wafer's features. Access holes were then cut out using 1 mm and 6 mm biopsy punches on both sides to form wells. This was also done carefully to avoid damaging the microchannel.

[0185] Bonding of PDMS / electrode array devices Next, the fabricated PDMS modules were bonded onto a custom MEA. This was achieved by placing the PDMS modules and the electrode surfaces to which they were bonded within a Zepto plasma system (Diener Electronic, Ebhausen, Germany). The surfaces were exposed to a 20% O2 plasma for 5 minutes under vacuum. The PDMS microchannels were carefully aligned with the ground electrode and recording electrode before being bonded onto the MEA. This was done manually using an inverted microscope (Figure 6D).

[0186] The following non-restrictive comments are provided regarding the use and verification of the device. I. Pancreatic Islets Pancreatic islets were isolated from the provided pancreas using enzymatic and mechanical methods, with the final step being the purification of the islets isolated from the exocrine tissue (Matsumoto et al. (2007)).

[0187] II. Electrophysiological Solutions A culture medium containing 7.3 mM glucose was used as the starting condition for the electrophysiological experiments. A culture medium supplemented with an additional 10 mM glucose (total 17.3 mM) was used as the high-glucose solution. The solution was also optimized to provide a "cleaner" electrophysiological record by using a pancreatic islet solution (Kindmark et al. (1994)) containing 138 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 5 mM CaCl2, HEPES, and pH 7.4. The pancreatic islet solution was supplemented with 3 mM and 15 mM glucose for the low (control) glucose condition and the high-glucose condition, respectively.

[0188] III. Electrophysiological Setup Electric field potentials were recorded using an Axon CNS Digidata 1440A digitizer (Molecular Devices, California, USA), a 16-channel microelectrode amplifier, Model 3600 (AM Systems, Inc., Washington, USA), and Clampex 10.7 software (Molecular Devices, California, USA). Initially, a 20-minute protocol was used to record the electrical activity of the pancreatic islets. This protocol was extended to 30 minutes. The electrical activity signals were pre-amplified using an x10 headstage (Omnetics, Minnesota, USA). Recording settings for the high-pass and low-pass filters were 3 Hz and 5 kHz, respectively. The gain was set to x2000, the GND setting was selected, and a notch filter was turned on to eliminate line-related noise at 50 / 60 Hz.

[0189] IV. Electrophysiological Protocols Pancreatic islets were used for 1–6 day experiments of culture in culture medium. 200 μl of sample from a 5 mL aliquot was transferred to the inlet channel of a microfluidic channel, and the position of the pancreatic islets relative to the electrode within the microfluidic channel was examined. 7.3 mM culture medium was used for initial loading. This prevented the pancreatic islets from adhering to the wall of the microfluidic channel. For 3 mM–15 mM glucose experiments, after adding to the MEA device, the remaining culture medium was removed and replaced with 200 μl of 3 mM glucose pancreatic islet solution. Once positioning was confirmed (via an inverted optical microscope), the pancreatic islets were cultured in the solution at room temperature for 10 minutes and then recorded. The MEA / microfluidic device was connected and placed under an inverted microscope, and after recording for 5 and 10 minutes, 17.3 mM glucose culture medium and 15 mM glucose pancreatic islet solution were added, respectively. The remaining starting solution was removed (via a micropipette) from the tip of the inlet pipette, and the high-concentration glucose solution was added via syringe. Positioning was reconfirmed using an inverted microscope, and recording continued for 15 or 20 minutes. Pancreatic islets from Batch 3 were evaluated using a 5.5 mM–15.5 mM culture medium protocol, with recordings made for 5 minutes before and after glucose addition. A total of 7 recordings were obtained from Batch 1, 11 from Batch 2, and 19 from Batch 3. Of these recordings, 11 were obtained using the 7.3 mM–17.3 mM glucose protocol, 8 using the 3 mM–15 mM protocol, 14 using the 5.5 mM–15.5 mM protocol, and 5 treated with tolbutamide (50 μM). Here, glucose concentrations of 3 mM, 5.5 mM, and 7.3 mM, as well as tolbutamide, were used as controls. 15 mM, 15.5 mM, and 17.3 mM glucose were treated as high concentrations. After each experiment, the MEA / microfluidic device was washed with deionized water before starting the repeat experiment. All experiments were conducted at room temperature (20°C).

[0190] V. Signal amplification The Model 3600 amplifier was purchased from AM Systems (Washington, USA) at a fraction of the cost of a commercially available array amplifier. It had 16-channel capability and 11 gain settings ranging from x2 to x20000, eight low-pass filters in the 100Hz to 20kHz range, eight high-pass filters in the 0.3Hz to 500Hz range, and a notch filter per channel (50Hz or 60Hz). The signal was pre-amplified by an x10 headstage (Omnetics (Minnesota, USA)) located next to the array, and the data was then passed to the Model 3600 amplifier. A device for flexibly holding the MEA and interfaced it to the preamplifier headstage was fabricated in the electronics workshop. This consisted of a retaining plate and a top plate, the top plate containing a spring pin (PD8 JS-2.2: Coda-Systems (Essex, UK)) with a 1 mm tip diameter and a 0.45 mm working stroke, used to interface with the electrode contact pads of the MEA. With the cooperation of the Electronics Workshop, a total of three recording devices (RD1, RD2, and RD3) were fabricated, each of which had a different arrangement of spring pins and connections from the device to the amplifier to enable recording from all 59 electrodes of a commercially available MEA, and to enable recording and stimulation when using a custom MEA. The pins were connected to the preamplifier head stage via nanostrip connectors (NPD-18-WD-18.0-C-GS: Omnetics). Each cable was soldered to one pin, with the exception of the ground cable and x10Ref cable which were soldered together on a single ground pin.

[0191] VI. Digitalization of Signals The analog signal from the amplifier was digitized using a digital data acquisition system (PCle-6343: National Instruments (Texas, USA)). The digital signal was displayed in real time using Clampex (Molecular Devices (California, USA)). All 16 channels could be displayed simultaneously on the Clampex.

[0192] VII.RMS quantification method The sensitivity of the root mean square variance (RMS) of the signal to detect changes in electrical response was validated by calculating the RMS of three separate records of pancreatic islets in glucose protocols of 7.3 mM–17.3 mM, 3 mM–15 mM, and 5.5 mM–15.5 mM for batches 1, 2, and 3, respectively. RMS was calculated at one-minute intervals throughout the entire recording and graphed with the recording to determine whether the RMS changes were synchronized with changes in electrical activity. RMS measurements were obtained using the power spectral function of Clampfit (version 10.7), with the window set to no signal change (rectangular), the output set to the average spectral segment, the length set to maximum, the spectral resolution (spectral bin width) set to 0.038147 Hz, and the RMS measurements and plots were set to exclude the first spectral bin (default settings). All RMS measurements of electrical response were in microvolts (μV) (Figure 7).

[0193] According to one embodiment, the combination of a microfluidic channel design and a trap or restrictor is incorporated into a printed microelectrode array as well as input and output chambers. The channel includes a microfluidic bypass, which allows the passage of solution once the islets are engaged within the trap. This flow-through system allows for rapid replacement of a normal storage solution (5.5 mM glucose) with a solution containing a higher concentration (15.5 mM glucose). The change in glucose concentration induces a measurable electrical change in the islets, which is detected by a reader. This activation pattern is observed only in fully functional islets. Multiple channels (1, 8, and 16 in our current device, but potentially up to 64 in future developments) allow for rapid determination of the percentage of functional islets in a sample. The device may enable rapid, accurate, and label-free assessment of the health of the islets. This would be deployed to objectively assess islet viability and to enable the use of pancreases with a small number of isolated islets in surgery, despite longer ischemic times. This will allow more material to be used for clinical transplants, enabling more patients to receive life-saving transplants and reducing the number and time on waiting lists. In addition, since there is an urgent need to improve methods for maintaining, sorting, and quality control of pancreatic islets, such devices will also contribute to increased efficiency at all levels.

[0194] In the embodiment, islet transplantation is performed in organ transplant patients with type 1 diabetes using islets isolated from the donor pancreas, and the field of stem cell-derived human islets has become a focus of vigorous research. This is a life-saving procedure that stabilizes blood glucose control and even leads to insulin independence. However, transplant outcomes may be closely related to the number of islets and their viability. The transplantation process is a multi-step procedure involving pancreatic procurement, tissue dissection, islet purification, cell culture, and islet transplantation into the organ transplant patient's liver via the hepatic portal vein. At each stage, islets are lost (mainly by ischemia). Since less than 50% of the islets obtained from a single pancreas are isolated (less than 500,000 islets), and it is estimated that less than 50% of these engraft in the liver, organ transplant patients require islets from 2-3 donor pancreases. There is no rapid method to establish the quality of islets in a sample before transplantation. The device provides a core technology for obtaining a rapid estimate (%) of functional islets in a sample. This would meet the need by measuring the electrical activity of pancreatic islets as a surrogate indicator of islet health. The design of the microfluidic delivery system and electrical interface (electrode array) together can provide a "hands-free" module for measuring the electrical function of pancreatic islets. Specifically, when an islet sample is pipetted into a 200 μL holder, the islets enter the system and are captured onto the electrodes by capillary action and gravity-driven flow. The specific design of the channel means that a further 200 μL solution containing elevated glucose can be pipetted into the delivery system to elicit an electrical response from viable islets. The specific combination of electrodes in the microfluidic module and a flow bypass system means that after a measurement is performed, the sample can be washed away and the measurement can be repeated with another sample.

[0195] The above description of specific embodiments is for illustrative purposes only. Those skilled in the art will understand that modifications of the described embodiments can be made without departing from the scope of the invention.

[0196] References 1. Alassaf, A.; Ishahak, M.; Bowles, A.; Agarwal, A. (2020) Microelectrode Array based Functional Testing of Pancreatic Islet Cells. Micromachines 2020, 11, 507. https: / / doi.org / 10.3390 / mi11050507 2. Barnett MJ, McGhee-Wilson D, Shapiro AMJ & Lakey JRT (2004). Variation in human islet viability based on different membrane integrity stains. Object Transplant 13, 481-488. 3. Benomar K, Chetboun M, Espiard S, Jannin A, Le Mapihan K, Gmyr V, Caiazzo R, Torres F, Raverdy V, Bonner C, D’Herbomez M, Pigny P, Noel C, Kerr-Conte J, Pattou F & Vantyghem MC (2018). Purity of islet preparations and 5-year metabolic outcome of allogenic islet transplantation. Am J Transplant 18, 945-951. 4. Boyd V, Cholewa O & Papas K (2008). Limitations in the Use of Fluorescein Diacetate / Propidium Iodide (FDA / PI) and Object Permeable Nucleic Acid Stains for Viability Measurements of Isolated Islets of Langerhans. Curr Trends Biotechnol Pharm 2, 66-84. 5. Brooks AM et al. (2013). Attainment of metabolic goals in the integrated UK islet transplant program with locally isolated and transported preparations. Am J Transplant 13, 3236-3243. 6. Bunnett J & Counter C (2019). Isolation Statistics. NHS BLOOD Transpl ORGAN DONATION Transplant Dir PANCREAS Advis Gr 1-7. Available at: moz-extension: / / 92fcdea0-1508-254e-b5f0-5bbad9269de7 / enhanced-reader.html?openApp&pdf=https%3A%2F%2Fnhsbtdbe.blob.core.windows.net%2Fumbraco-assets-corp%2F17532%2Fisolation-statistics.pdf [Accessed May 24, 2022]. 7. CITR Coordinating Centre (2015). Scientific Summary of the Collaborative Islet Transplant Registry (CITR) 2015 (Tenth) Annual Report BACKGROUND AND PURPOSE. CITR Coordinating Center The Emmes Corporation, Rockville, MD. Available at: https: / / citregistry.org / system / files / 10AR_Scientific_Summary.pdf[Accessed May 24, 2022]. 8. Cornateanu SM, O’Neill S, Dholakia S, Counter CJ, Sherif AE, Casey JJ, Friend P & Oniscu GC (2021). Pancreas utilization rates in the UK - an 11-year analysis. Transpl Int 34, 1306-1318. 9. Dufer M (2012). Determination of Beta-Object Function: Ion Channel Function in Beta Objects. In Animal Models in Diabetes Research, pp. 203-217. Humana Press. Available at: https: / / pubmed.ncbi.nlm.nih.gov / 22893409 / [Accessed May 23, 2022]. 10. Forbes S, McGowan NWA, Duncan K, Anderson D, Barclay J, Mitchell D, Docherty K, Turner D, Campbell JDM & Casey JJ (2015). Islet transplantation from a nationally funded UK centre reaches socially deprived groups and improves metabolic outcomes. Diabetologia 58, 1300-1308. 11. JDRF Editors (2022). Facts and figures about type 1 diabetes - JDRF, the type 1 diabetes charity. JDRF. Available at: https: / / jdrf.org.uk / information-support / about-type-1-diabetes / facts-and-figures / [Accessed May 23, 2022]. 12. Kindmark H, Kohler M, Arkhammar P, Efendic S, Larsson O, Linder S, Nilsson T & Berggren PO (1994). Oscillations in cytoplasmic free calcium concentration in human pancreatic islets from subjects with normal and impaired glucose tolerance. Diabetologia 37, 1121-1131. 13. Matsumoto S, Noguchi H, Naziruddin B, Onaca N, Jackson A, Hatanaka N, Okitsu T, Kobayashi N, Klintmalm G & Levy M (2007). Improvement of Pancreatic Islet Object Isolation for Transplantation. Baylor Univ Med Cent Proc 20, 357-362. 14. Pfeiffer T, Kraushaar U, Dufer M, Schonecker S, Haspel D, Gunther E, Drews G & Krippeit-Drews P (2011). Rapid functional evaluation of beta-objects by extraobjectular recording of membrane potential oscillations with microelectrode arrays. Pflugers Arch Eur J Physiol 462, 835-840. 15. Schonecker S, Kraushaar U, Dufer M, Sahr A, Hardtner C, Guenther E, Walther R, Lendeckel U, Barthlen W, Krippeit-Drews P & Drews G (2014). Long-term culture and functionality of pancreatic islets monitored using microelectrode arrays. Integr Biol (United Kingdom) 6, 540-544. 16.Schonecker S, Kraushaar U, Guenther E, Gerst F, Ullrich S, Haring HU, Konigsrainer A, Barthlen W, Drews G & Krippeit-Drews P (2015). Human islets exhibit electrical activity on microelectrode arrays (MEA). Exp Clin Endocrinol Diabetes 123, 296-298.

Claims

1. A microfluidic device, It comprises a microfluidic module, and the microfluidic module is Multiple fluid paths between the input and output, The microfluidic module includes a plurality of limiting units, each limiting unit provided on each of the plurality of fluid paths, and each limiting unit is configured to capture or at least restrict the movement of one or more objects, e.g., one or more tissues and / or multicellular objects, in the fluid introduced along each fluid path in each sensing region, and the microfluidic module further includes, The system includes at least one further fluid path between the input and the further output, the at least one further fluid path being in fluid communication with the plurality of limiting units, the further fluid path including a delivery portion between the input and the plurality of limiting units for enabling the delivery of further fluid to one or more captured and / or restricted objects, and an output portion for enabling the removal of the further fluid from the further output, The aforementioned device further, It is equipped with an electronic detection module, and the electronic detection module is A microfluidic device comprising at least one sensing element arranged to detect one or more signals from each of the sensing regions of the plurality of limiting sections.

2. The device according to claim 1, wherein the object comprises at least one of an organoid, spheroid, tissue spheroid, pancreatic islet, cell, or tissue object.

3. The device according to any one of the preceding claims, wherein the object comprises tissue spheroids, optionally pancreatic islets derived from the pancreas.

4. The device according to any one of the preceding claims, wherein the further fluid path at least partially overlaps with the plurality of fluid paths in the delivery portion, and the output portion is spatially separated from the plurality of fluid paths.

5. The output portion of the further fluid path is downstream from the delivery portion and the plurality of limiting portions of the device according to any one of the preceding claims.

6. The device according to any one of the preceding claims, wherein the further fluid path comprises a path for flushing and / or removing at least fluid from the device via the further output.

7. It has multiple channels, and the multiple channels are A first channel is provided between the input and the further output, the first channel defines the further fluid path between the input and the further output, and the plurality of channels further, The device according to any one of the preceding claims, comprising the plurality of limiting units and a second channel coupled to the output, wherein at least a portion of the first channel and at least a portion of the second channel are coupled to the first channel via the plurality of limiting units such that a plurality of fluid paths are defined between the input and the output.

8. The device according to any one of the preceding claims, wherein the first channel and the second channel are sized to allow fluid flow of the object, and the restricting portion has an opening between the first channel and the second channel that is sized to prevent the object from passing through.

9. The device according to any one of the preceding claims, wherein the plurality of limiting units include five or more, optionally at least ten, optionally at least fifteen, optionally at least sixty, or optionally at least 100 limiting units.

10. The device according to any one of the preceding claims, wherein the plurality of limiting sections are arranged in the overlapping portions of the plurality of fluid paths and the further fluid paths to enable the continuous delivery of objects to the plurality of limiting sections such that when one limiting section is blocked by an object, further objects in the fluid proceed to subsequent limiting sections and / or toward the further output.

11. The device according to any one of the preceding claims, wherein the at least one sensing element is optionally provided on a layer below the plurality of restricting portions such that at least partially restricted and / or captured objects come into contact with the at least one sensing element.

12. The device according to any one of the preceding claims, wherein the at least one sensing element comprises a plurality of sensing elements each having one or more sensing elements and at least one reference electrode for each limiting portion, and / or the at least one sensing element is configured to detect an electrophysiological signal and / or signal in response to the electrophysiological activity of an object.

13. The detection module comprises one or more stimulating elements, e.g., stimulating electrodes, configured to generate an electronic stimulus, e.g., a stimulating signal, and one or more sensing elements, e.g., sensing electrodes, configured to detect a response to the electronic stimulus, wherein optionally, the one or more stimulating elements and / or sensing elements are positioned to stimulate and / or detect activity within the sensing region, according to any one of the preceding claims.

14. The device according to any one of the preceding claims, wherein the at least one sensing element is provided as part of an electrode array, aligned with and / or located near the at least one limiting portion.

15. The microfluidic device according to any one of the preceding claims, further comprising a reference sensing element, optionally provided in or across at least a portion of the plurality of fluid paths and / or at least a portion of the further fluid paths.

16. The device according to any one of the preceding claims, wherein each of the limiting portions comprises an opening having a cross-sectional area at least 50%, 60%, 70%, 80%, 90%, or 95% smaller than the diameter of the one or more objects to be targeted.

17. The device according to any one of the preceding claims, wherein the plurality of fluid paths and the at least one further fluid path are formed by channels having a cross-sectional area at least 50%, 60%, 70%, 80%, 90%, or 95% larger than the diameter of the one or more objects in question.

18. The microfluidic device according to any one of the preceding claims, wherein the plurality of fluid paths and the further fluid paths are formed by a plurality of channels, and at least one of the widths and / or heights of at least one channel is selected from a range that increases or decreases the fluid velocity.

19. An apparatus comprising the device according to any one of claims 1 to 18, wherein the apparatus includes processing resources, and the processing resources are The system receives a sensor signal and / or data representing the sensor signal from at least one of the detection elements. The data and / or signals are processed to determine at least one of the following: electrical activity, viability, function, and / or response to stimulation, and optionally, A device configured to deliver electrical stimulation through one or more stimulating electrodes.

20. The processing resource is configured to determine at least one of the following, and at least one of the following is a) A measure of electrical activity in response to electrical stimulation and / or further fluids, wherein the further fluids include chemicals and / or drugs, such as glucose solutions, and at least one of the following is further: b) Some viable and / or functional tissues and / or multicellular objects in the sample, c) The apparatus according to claim 19, comprising performing a diagnosis and / or display of viability based on detected electrical activity in the plurality of limiting and / or sensing regions, for example, the diagnosis and / or display of viability may be based on electrical activity in response to exposure of the object to further substances including chemicals and / or drugs, such as glucose, or to an electrical stimulus.

21. A method for analyzing multiple objects, for example, multiple tissues and / or multicellular objects, using the device described in claims 1 to 19, wherein the method is: The method includes the step of providing a plurality of objects in a fluid to the input of the microfluidic device, the fluid initially flowing through the device along the plurality of fluid paths until the plurality of objects reach the plurality of limiting parts and are captured and / or restricted by the limiting parts, and the method further, A method comprising the step of detecting at least one sensor signal for the plurality of objects using the at least one detection element.

22. The method according to claim 21, further comprising the step of delivering further fluid through the input to the plurality of objects trapped or at least restricted within the plurality of restrictors via at least one further fluid path.

23. The method according to claim 21 or 22, further comprising the step of delivering electrical stimulation to the plurality of objects that are trapped or at least restricted by the plurality of restricting portions.

24. The method according to claims 21 to 23, wherein the one or more objects originate from the pancreas, for example, islets or islets of Langehans, and / or the one or more objects move by gravity and / or capillary action to and / or beyond at least one limiting portion.

25. The aforementioned further fluid is a) A washing solution for removing any solution via the further output, b) Glucose solution and c) One or more drug and / or cell and / or substance activity inhibitors, d) One or more molecules for labeling the object, e) a concentration higher than that of the fluid containing the plurality of objects, the method according to claims 21 to 24, comprising at least one of these.

26. The method according to any one of claims 21 to 25, further comprising the step of isolating an output electrical signal from a captured object for the plurality of limiting portions of the microfluidic device.

27. The method according to any one of claims 21 to 26, further comprising the step of processing the sensor signal and / or data representing the sensor signal to evaluate at least one of the following: the electrical activity of a cell, the potential generated extracellularly, the electric field potential, the viability, the function, and / or the response to a stimulus.

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