Device for capturing tissue and / or multicellular objects

Abstract

A microfluidic device comprising: a microfluidic module comprising a plurality of fluid paths between an input and an output; and a plurality of restrictions, where each restriction is disposed on a respective fluid path of the plurality of fluid paths, where each restriction is configured to: at a respective sensing region, sense the fluid path of the fluid path; capturing or at least limiting the movement of one or more objects (e.g., one or more tissues and / or multicellular objects) in the fluid introduced along their respective fluid paths; and at least one further fluid path between the input portion and the further output portion, where the at least one further fluid path is in fluid communication with the plurality of restrictions, and where the further fluid path comprises a delivery portion and an output portion, where the delivery portion is in fluid communication with the plurality of restrictions. A delivery portion between the input portion and the plurality of restrictions to allow delivery of additional fluid to the captured and / or restricted one or more objects, and an output portion allowing removal of additional fluid from the additional output portion; wherein the apparatus further comprises an electronic sensing module, the electronic sensing module comprising at least one sensing element, the at least one sensing element being arranged to sense one or more signals from respective sensing areas of the plurality of restrictions.

Description

Technical Field

[0001] This disclosure relates to a microfluidic device, particularly an apparatus for capturing and testing multiple objects (e.g., one or more tissues and / or multicellular objects). A method is provided for using said apparatus to determine, for example, the mass and / or function of cells, tissues, or objects. Background Technology

[0002] Type 1 diabetes (T1D) is an autoimmune disease affecting 400,000 people in the UK (JDRF Editors, 2022). This condition is characterized by the loss of functional beta cells, the majority of cells in a cluster of cells called islets of Langerhans located in the pancreas. An islet comprises approximately 1500 cells, 74% of which are beta cells. These beta cells normally secrete insulin upon glucose stimulation, but this function is lost in T1D, resulting in uncontrolled and / or high blood glucose levels. While treatment with injectable synthetic insulin can help control blood glucose levels and is the primary treatment, feedback control is absent, and a common side effect of insulin therapy is hypoglycemia. Recurrent episodes of hypoglycemia can lead to impaired awareness of hypoglycemia, making it difficult for individuals to recognize low blood glucose levels, which can result in significant morbidity and mortality. Islet transplantation via cadaveric donors for the treatment of T1D has been shown to be effective in improving glucose control. Transplantation can reduce the number of severe hypoglycemic events and is an effective treatment for restoring awareness of hypoglycemia. In rare 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 pancreas for islets is a major limiting factor; in the UK, only about 100 donor pancreas were available in 2019, many of which were unsuitable or produced poor quality and quantity of islets (Cornateanu et al., 2021). The fact that each recipient typically requires islets from 2 to 3 donors to significantly impact their glycemic control further complicates the situation.

[0003] Islet count, sample purity, and islet viability are all important parameters for meeting clinical release criteria before transplantation (Brooks et al., 2013; Benomar et al., 2018). In the UK, islet viability must be greater than 70% for islet preparation to be transplanted. Overestimating islet viability may result in transplanting a relatively large number of non-viable islets and an insufficient number of islets, while underestimating islet viability may result in the discarding of islets that have survived in other ways. These criteria protect recipients by ensuring that only pancreas with a large number of healthy islets is transplanted.

[0004] However, this has resulted in a low clinical conversion rate, which was 28% in 2019 (Bunnett J and Counter C, 2019). Even so, within the Collaborative Islet Transplant Registry, approximately 70% of islet transplant recipients are insulin-independent one year post-transplant, and this figure drops to approximately 40% five years post-transplant (CITR Coordinating Centre, 2015). Therefore, while insulin independence in T1D is achievable, the trend in the rate of decline several years post-transplant indicates the importance of islet quality and quantity for long-term positive outcomes. In conclusion, the decline in clinical outcomes after transplantation, along with the low percentage of donor pancreas meeting release criteria, points to an urgent need for objective islet assessment, with the aim of improving the quantity and quality of islets available for transplantation.

[0005] Currently, fluorescein diacetate and propidium iodide staining for membrane integrity (Barnett et al., 2004) is the preferred method for assessing survival. While this method is rapid and easy to apply, it suffers from problems due to the subjective interpretation of results, leading to frequent overestimations of survival (Boyd et al., 2008). There is increasing evidence that dying, inflamed islets can adversely affect the entire islet graft, impairing the function of initially healthy islets. Therefore, developing a robust, real-time, and more objective method for assessing islet survival prior to transplantation is a pressing priority.

[0006] Measuring the electrophysiological response of β-cells in the islets to glucose is an attractive alternative assessment method because the electrical response generated 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 survival. Determining β-cell electrical activity typically involves conventional electrophysiological methods such as single-cell and / or whole-cell voltage clamping techniques or intracellular electrodes (Pfeiffer et al., 2011; Düfer, 2012). These techniques are very time-consuming and require expert-level single-cell manipulation and interpretation. On the other hand, extracellular recording using aspiration electrodes and microelectrode arrays (MEAs) can detect islet electrical activity with minimal tissue micromanipulation. In fact, complete mouse and human islet electrical activity has been demonstrated in single and multiple islets, suggesting that this approach may be a viable method for assessing islet survival (Pfeiffer et al., 2011; Schnecker et al., 2014, 2015), as the lack of or abnormal electrical activity is an indicator of GSIS dysfunction.

[0007] Therefore, although previous studies have shown the electrophysiological response of islet isolation, these methods involve specialized equipment, knowledge, and the ability to manually manipulate individual islets and interpret the results.

[0008] One of the purposes of this disclosure is to eliminate and / or mitigate at least one of the aforementioned disadvantages. Summary of the Invention

[0009] In a first aspect, a microfluidic device is provided, comprising: a microfluidic module including a plurality of fluid paths between an input and an output; and a plurality of restraints, each restraint disposed on a corresponding fluid path of the plurality of fluid paths, wherein each restraint is configured to: capture or at least restrict movement of one or more objects (e.g., one or more tissues and / or multicellular objects) in fluid introduced along its corresponding fluid path at a corresponding sensing region; and at least one additional fluid path between the input and the additional output, wherein the at least one additional fluid path is in fluid communication with the plurality of restraints, and wherein the additional fluid path includes a delivery portion and an output portion, the delivery portion being between the input and the plurality of restraints to allow delivery of additional fluid to the captured and / or restrained one or more objects, and the output portion allowing removal of the additional fluid from the additional output. The device may further include an electronic sensing module including: at least one sensing element arranged to sense one or more signals from corresponding sensing regions of the plurality of restraints.

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

[0011] The sensing module may include one or more stimulating elements and one or more sensing elements, the stimulating elements being configured to generate electronic stimulation, and the sensing elements being configured to measure the sensed signal (optionally, a potential). The stimulating elements may be configured to generate electronic stimulation, and the sensing elements may be configured to sense a response to the stimulation.

[0012] The object may include at least one of the following: organoids, cellular spheroids, tissue spheroids, and / or pancreatic islets. The object may include cellular objects and / or tissue objects.

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

[0014] The object may include a tissue spheroid derived from the pancreas. The tissue and / or multicellular object may include a group of objects, such as cells in an organ having at least one functional, structural, or biological property.

[0015] The additional fluid path may at least partially overlap with the plurality of fluid paths at the delivery section, and wherein the output section is spatially separated from the plurality of fluid paths.

[0016] The output portion of the additional fluid path may be downstream of the delivery portion and the plurality of limiting elements.

[0017] Additional fluid paths can provide a route for flushing and / or removing at least fluid and objects from the device via additional outputs.

[0018] The first channel may include a fluid and / or object transport section. The first channel may include a fluid and / or object flushing section. Multiple restraints may be provided at or on the fluid transport section and / or object transport section.

[0019] The plurality of restraints may be configured to be adjacent to and in fluid communication with the at least one additional fluid path. The at least one additional fluid path may be arranged to expose the captured and / or restrained object to the additional fluid. The delivery portion may be a shared input portion and may form part of the first channel. The output portion may include a common output portion.

[0020] The device may include a plurality of channels, the plurality of channels including: a first channel between the input and the additional output, the first channel defining the additional fluid path between the input and the additional output; and a second channel connected to the plurality of limiting members and the output, wherein the second channel is connected to the first channel via the plurality of limiting members 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 dimensions of the first and second channels can be designed to allow fluid flow of the object. The restraint may include an opening between the first and second channels, the opening being sized to prevent the object from passing through.

[0022] The input section may include an inlet. The output section may include an outlet. The additional output section may include an additional outlet. Fluid can flow from the input section to the additional output section at a higher flow rate when the limiting member is substantially blocked, compared to when the limiting member is substantially unblocked. The at least one limiting member may form part of a trap for capturing one or more objects (e.g., one or more tissues and / or objects). The objects may be cellular and / or tissue objects. The limiting member may be sized to allow fluid flow through it and prevent objects from passing through the limiting member.

[0023] The at least one sensing element may include electrodes. The at least one sensing element may include a passive high-impedance electrode to measure the field potential relative to a passive or grounded low-impedance reference electrode. The at least one sensing element may be disposed at a sensing region. The device may include hybrid microfluidics and microelectronic devices.

[0024] The microelectrode array may include one or more stimulating electrodes for providing stimulation to one or more sensing regions, and / or a captured object, and / or one or more objects within the sensing regions. The one or more stimulating electrodes may include pairs of stimulating electrodes for providing stimulation to the sensing regions (optionally objects within the sensing regions). The one or more stimulating electrodes may include electrode pairs utilizing a low-impedance reference electrode.

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

[0026] The sensing module may include at least one passive sensing electrode element, and, for example, at least one stimulating electrode and a reference electrode. The stimulating electrode and the reference electrode may form one or more electrode pairs. The electrode pairs may be arranged on either side of the sensing region, such that the sensing region is positioned between the stimulating electrode and the sensing electrode. The at least one sensing electrode may be configured to sense a response in the object to electrical stimulation delivered from the stimulating electrode.

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

[0028] The plurality of restrictors may be arranged at the overlap of the plurality of fluid paths and the other fluid path to allow objects to be sequentially delivered to the restrictors, such that when a restrictor is blocked by an object, another object in the fluid advances to the subsequent restrictor and / or toward the other output.

[0029] The plurality of fluid paths and additional fluid paths may be formed by channels whose dimensions are designed to allow the flow of one or more objects.

[0030] The at least one sensing element may be disposed on a layer below the plurality of restraints, optionally such that the object at least partially restrained and / or captured comes into contact with the at least one sensing element.

[0031] The at least one sensing element may include a plurality of sensing elements, the plurality of sensing elements including one or more sensing elements, the one or more sensing elements being used for each limiting element and at least one reference electrode.

[0032] The sensing element can be configured to sense electrophysiological signals and / or biological signals in response to the electrophysiological activity of an object. The electrophysiological or other activity of the object can be responsive to chemical stimulation and / or chemical stimuli provided at the input. The electrophysiological or other activity of the object can be responsive to chemical stimulation and / or chemical stimuli provided at the input and flushed by the system. The electrophysiological or other activity of the object can be responsive to provided electrical stimulation, such as electrical stimulation provided to the object from one or more stimulating elements or electrodes.

[0033] The at least one sensing element may be configured to be aligned with and / or positioned adjacent to a portion of the at least one limiting member in an electrode array. The electrode array may be aligned to contact the one or more objects. The at least one sensing element may include one or more sensing or recording electrodes. The sensing electrode may be a passive electrode. The sensing electrode may include an exposed portion that contacts the object. The diameter of the exposed portion may range from 10 micrometers to 100 micrometers, and more preferably from 20 micrometers to 60 micrometers. The diameter of the exposed portion may depend on the material used.

[0034] The sensing module may include multiple conductor tracks. These conductor tracks are, for example, connected to corresponding components (e.g., electrode pads) or components forming multiple electrodes. The tracks may lead to the corresponding electrode pads. The tracks may be conductive. The tracks may be electrically insulated from the fluid paths and / or channels of the microfluidic device. In use, the tracks may be electrically insulated from the fluid contents of the microfluidic device. The pads and / or tracks may contain conductive materials (e.g., titanium or gold deposition). The tracks may be insulated using insulating materials (e.g., silicon oxide).

[0035] At least one pair of stimulating electrodes may be configured in the electrode array to be aligned with at least one limiting element and the passive recording electrode and / or to be located adjacent to at least one limiting element and the passive recording electrode. The electrode array may be substantially planar.

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

[0037] The reference sensing element can serve as a reference sensing element for at least one (optionally multiple) sensing elements. At least a portion of the reference sensing element can be disposed at or overlap with the plurality of fluid paths and / or additional fluid paths. The plurality of sensing electrodes can be aligned with the plurality of limiting members. The plurality of sensing electrodes can be provided in a substantially linear arrangement. The spacing between subsequent sensing electrodes can correspond to the spacing between the plurality of limiting members. The sensing elements can be arranged such that each limiting member is substantially disposed between the corresponding sensing element and reference element. The exposed portion of the reference electrode can be at least a portion, optionally all or entirely, of an electrode pad providing a passive low-impedance voltage reference. The exposed portion of the reference electrode can include passive low-impedance components.

[0038] Each of the limiting members may include an opening whose cross-sectional area is at least 50%, 60%, 70%, 80%, 90%, or 95% smaller than the diameter of one or more objects of interest.

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

[0040] The dimensions of the object may be, for example, a width and / or height in the range of 150 micrometers to 300 micrometers.

[0041] The plurality of limiting elements may include widths smaller than the width of the object of interest. The limiting elements may include widths of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the width of the object of interest.

[0042] The plurality of limiting elements may have a width between 10 micrometers and 75 micrometers, optionally between 20 micrometers and 40 micrometers. The width of the channel may be between 300 micrometers and 1000 micrometers, optionally between 400 micrometers and 500 micrometers, and further optionally 450 micrometers. The height of the channel may be between 200 micrometers and 1000 micrometers, optionally between 200 micrometers and 300 micrometers, and further optionally 250 micrometers.

[0043] The plurality of fluid paths and the at least one additional fluid path may be formed by channels whose width is at least 50%, 60%, 70%, 80%, 90%, or 95% greater than the width of the one or more objects. The plurality of fluid paths and the at least one additional fluid path may be formed by channels whose height is at least 50%, 60%, 70%, 80%, 90%, or 95% greater than the height of the one or more objects.

[0044] The width and / or size of the object of interest may include the average width and / or size of such an object, or its typical width and / or size.

[0045] The plurality of fluid paths and the additional fluid paths may be formed by a plurality of channels. At least one of the width and / or height of at least one channel may be selected from a range, thereby increasing or decreasing the fluid flow rate.

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

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

[0048] Data and / or signals can be processed to determine the percentage of surviving tissue and / or objects in a sample. Determining at least one of electrical activity, survival rate, function, and / or response to stimuli may include generating and storing data representing electrical activity, function, and / or response to stimuli.

[0049] The processor can be configured to determine at least one of the following:

[0050] a) Measurement of electrical activity in response to additional fluid and / or direct electrical stimulation, optionally wherein the additional fluid contains chemicals and / or drugs (e.g., glucose solution), and optionally wherein the electrical stimulation is provided by one or more stimulating elements or electrodes;

[0051] b) The number of multiple surviving objects, and / or functional tissue objects, and / or multicellular objects in the sample.

[0052] c) Performing diagnostic and / or survival indications based on electrical activity sensed at the plurality of restraints and / or sensing areas, for example, wherein the diagnostic and / or survival indications may be based on electrical activity from exposure of the object to an additional substance or electrical stimulation, the additional substance optionally comprising chemicals and / or drugs, such as glucose. The chemicals and / or drugs may include the chemicals and / or drugs to be tested. The response may be responsive to metabolic stimulation, such as glucose and / or electrical stimulation.

[0053] According to a second aspect, a method is provided for providing one or more objects of interest to an apparatus according to a first aspect, the method comprising providing one or more objects in a fluid to an input portion of the apparatus, wherein the fluid initially flows through the apparatus until the one or more objects reach the plurality of restraints and are captured and / or at least restrained at the plurality of restraints.

[0054] According to a third aspect, a method is provided for analyzing multiple objects (e.g., multiple tissue objects and / or multicellular objects) using the apparatus according to the first aspect, the method comprising: providing the multiple objects in a fluid to the input portion of the microfluidic device, wherein the fluid initially flows through the device along the multiple fluid paths until the multiple objects reach the multiple restraints and are captured and / or at least restrained by the restraints; and using the at least one sensing element to sense at least one sensor signal of the multiple objects.

[0055] The method may further include delivering additional fluid through the input to the plurality of objects that are captured or at least confined within the restraints via the at least one additional fluid path. The method may also optionally include applying electrical stimulation via one or more electrode pairs.

[0056] The at least one sensor signal can represent or indicate the biological activity of the plurality of objects. The at least one sensor signal may include one or more first sensor signals sensed before delivery of the additional solution and / or electrical stimulation and one or more additional sensor signals sensed after delivery of the additional solution and / or electrical stimulation. The one or more first sensor signals can represent or indicate the baseline electrophysiological activity of the plurality of objects. The additional signals can represent or indicate the electrophysiological activity of the plurality of objects. The at least one sensor signal can optionally be sensed for each constraint via a sensing element provided at each constraint, and further optionally, the at least one sensor signal can be sensed between the sensing element at each constraint and a reference electrode. The at least one sensor signal can be sensed relative to a reference electrode for each constraint.

[0057] The one or more objects may originate from the pancreas, for example, pancreatic islets, also known as Langerhans islets.

[0058] The one or more objects may be derived from other commonly available spherical objects, such as cardiac spheres, neural organoids, and other excitable object organoids.

[0059] The one or more objects may be moved to the at least one limiting member and / or moved beyond the at least one limiting member by gravity, volumetric flow, and / or capillary action.

[0060] The additional fluid may contain a washing solution for removing any solution and / or object via the additional output. The additional fluid may contain a glucose solution. The additional fluid may contain one or more drugs, and / or objects, or cell activity inhibitors. The additional fluid may contain one or more molecules for labeling the object. The additional fluid may contain a higher concentration than the fluid comprising the plurality of objects.

[0061] The method may further include isolating the output electrical signals from the captured object for multiple limiting elements of the microfluidic device. The method may include isolating the electrical signals of at least 15, optionally 30, or optionally 60 traps.

[0062] The method may further include: processing sensor signals and / or data representing sensor signals to assess at least one of cellular or object electrical activity, survival rate, function, and / or response to stimuli.

[0063] According to a fourth aspect, an apparatus is provided, the apparatus comprising the means of the first aspect and processing resources. The processing resources may correspond to the processor of the first aspect. The processing resources may be configured to receive sensor signals and / or data representing the sensor signals from the at least one sensing element;

[0064] The data and / or signals are processed to determine at least one of electrical activity, survival rate, function, and / or response to stimuli.

[0065] Data and / or signals can be processed to determine the percentage of surviving tissue and / or cells and / or objects in a sample. Determining at least one of electrical activity, survival rate, function, and / or response to stimuli may include generating and storing data representing electrical activity, function, and / or response to stimuli.

[0066] The processor may be configured to determine at least one of the following: a) a measurement of electrical activity in response to an additional fluid, optionally wherein the additional fluid comprises chemicals and / or drugs (e.g., glucose solution); b) the number of surviving and / or functional tissues and / or multicellular objects in the sample; c) performing a diagnostic and / or survival indication based on electrical activity sensed at the plurality of restraints and / or sensing areas, for example wherein the diagnostic and / or survival indication may be based on electrical activity in response to exposure of the object to an additional substance and / or direct electrical stimulation of the object, the additional substance optionally comprising chemicals and / or drugs (e.g., glucose).

[0067] The device may also include a display and a user input unit.

[0068] In a fifth aspect, a microfluidic / microelectronic / multichannel device is provided, wherein at least one of the microfluidic modules comprises:

[0069] (a) A first channel for receiving fluid and including an inlet and an outlet, and including at least 10 traps disposed between the inlet and the outlet for capturing (multiple) tissue / organoid samples introduced into the first channel through the inlet, wherein the traps include a confinement sized to allow fluid flow through the confinement but not the one or more tissues / organoids, and the traps further include at least 10 electrodes for detecting electrophysiological signals of the one or more tissues captured in the traps; and

[0070] (b) A second channel, the second channel being used to receive fluid from the first channel after said / each constraint has been blocked by said one or more tissues / organoids, thereby preventing further fluid from flowing through said / each constraint. The second channel may also be used to remove fluid and / or (multiple) objects after analysis.

[0071] Although this disclosure is generally described in the context of pancreatic islets, this should not be construed as limiting. The apparatus described in this disclosure can be provided with any type of tissue, wherein the electrical response generated by a plurality of active cells and / or objects therein allows for assessment of their viability and / or function of interest (e.g., cardiac or neuronal tissue). In one embodiment, the one or more tissues may be derived from the pancreas. In a preferred embodiment, for example, the one or more tissues derived from pancreatic islets comprise thousands of 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 excitable changes in the membrane potential can be used to assess the viability and / or function of various types of excitable tissues.

[0072] The membrane potential refers to the voltage or potential difference across the cell membrane. This potential difference is caused by a hydrophobic membrane that separates charges, acting as both a capacitor and a resistor to allow charged ions to move across it. The plasma membrane ensures the structural integrity of the cell and physically separates the intracellular compartments from the extracellular environment. A potential gradient exists across the cell membrane due to the significant differences in ionic composition between the intracellular and extracellular compartments, due to selective permeability for certain ion species, and due to the insulating physical properties of the phospholipid bilayer that constitutes the cell membrane. In a steady state, the intracellular compartments of the cell carry a greater negative charge relative to the extracellular environment (relative to potassium ions), resulting in a membrane potential ranging from -10 to -80 mV (depending on the cell type). In excitable tissues (such as pancreatic islets, muscle, and neurons (including those of stem cell origin)), rapid changes in membrane potential (e.g., action potentials) can be triggered by electrical and / or chemical signals through ion channels with durations of milliseconds and amplitudes exceeding 100 mV, which alter ion permeability.

[0073] Cellular electrophysiological state and / or response refers to the cell's electrical properties or activities, which are typically reflected in the aforementioned changes in membrane potential. The charge-dependent electrical state or cellular activity of a cell is ionic, such as sodium ions (…). ), chloride ions ( ), potassium ions ( ), and calcium ions ( Ion inflow and efflux from cells are 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). For example, external electrodes, microelectrodes, or arrays of microelectrodes can be used to detect synchronized cellular electrical activity in single or multiple cells by measuring and / or detecting extracellular local field potentials (which are a fraction of actual potential changes (5 μV to 100 μV)). ​​Such methods are clinically used to measure brain potentials (EEG), cardiac field potentials (ECG), and muscle potentials (EMG) when many cells are operating synchronously.

[0074] Therefore, in alternative embodiments, this disclosure may relate to objects, organoids, or tissues derived from other organs, wherein it is meaningful to assess the function and / or response of the entire object through changes in extracellular local field potential. Not limited to the examples provided herein, alternative organs (meaning the tissues of the objects described in this method can be derived from said alternative organs) may include, for example, the heart, brain, intestine, liver, kidney, gallbladder, stomach, or skin, and endothelial tissue, as well as stem cell derivatives from multiple organs. Objects (or cells) derived from organs and tissues used according to this disclosure may include cultured cellular objects, particularly cultured mammalian material, for testing the object viability and / or function of such cultured objects. In some embodiments, the tissues of this disclosure may include a group of cells or cell clusters that may be provided to the microfluidic device. For example, such tissues may include multiple organoids (e.g., brain organoids, cardiac spheroids), or embryonic stem cells, or induced pluripotent stem cell (iPSC) derivatives. In some embodiments, the tissues may include cell clusters, object clusters, cell spheroid clusters, or object spheroid clusters. Therefore, those skilled in the art will also recognize that the dimensions of the first channel, the second channel, the trap, and / or the restraint can be adjusted accordingly based on the size of the tissue to be provided to the microfluidic device for testing, the size of the cell cluster, or the size of the cell group. In particular, at least the dimensions of the first channel and the trap(s) should be designed such that at least one tissue object can fit within the dimensions of the first channel and the trap(s).

[0075] The input section serves as an inlet route through which fluid can be supplied to the microfluidic module. In a preferred embodiment, the fluid, such as a liquid (e.g., cell culture medium, saline, buffer solution, etc.), comprises one or more tissue objects, which are supplied to the microfluidic module through the input section. The input section may include an input chamber through which fluid (such as fluid from a reservoir) is supplied to the channel of the microfluidic module. The fluid supplied to the input section ideally flows through the channel by gravity and capillary drive. The use of gravity or capillary flow ensures that the tissue object is manipulated in the gentlest possible way, which differs from prior art systems that may employ pumps or the like to drive or aspirate fluid and objects through the microfluidic device. The microfluidic module also includes an output section (such as an output channel, and / or a well / reservoir) through which the fluid that has flowed through the channel can be collected. In some embodiments, the microfluidic module of the microfluidic device is connected to the same input section. In some embodiments, the microfluidic module of the microfluidic device is connected to the same output section. One or more objects are conveyed by the fluid flow of the device to trap(s), which(s) block the fluid flow through the trap(s).

[0076] In one embodiment, the cross-sectional area of ​​the trap is at least 50%, 60%, 70%, 80%, 90%, or 95% larger than the cross-sectional area of ​​the one or more objects being tested. In one embodiment, the cross-sectional area of ​​the trap region is at least 50% larger than the cross-sectional area of ​​the one or more objects resulting in confinement. In a preferred embodiment, the maximum diameter of the cross-sectional area of ​​the trap and / or the confinement may be between 10 μm and 60 μm. The trap is adjacent to the confinement, which is sized such that the tissue cluster or one or more objects cannot pass through the confinement(s). The confinement preferably includes a cross-sectional area that prevents one or more objects from passing through the confinement and traps the object, thereby blocking the confinement and, after trapping the object, preventing fluid from flowing through the confinement or minimizing the fluid flowing through the confinement. For example, it is known in the art that mammalian tissues, clusters (such as islets of Langerhans and globularia of the heart) typically have diameters of 100 μm to 300 μm. Therefore, in one embodiment, the maximum diameter of the cross-sectional area of ​​the limiting member can 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 cross-sectional area of ​​the limiting member can be 40 ± 10 μm. In another embodiment, the dimensions of the channels(s) can be: a width of 450 ± 50 μm and / or a height of 450 ± 50 μm.

[0077] After the fluid flow through the restrictor is blocked by one or more of the objects (the objects provided to the fluid device), any fluid flowing through the device is primarily directed toward and through the second channel. It should be noted that fluid can flow through the second channel before and after the blockage of each restrictor by one or more of the tissue objects occurs. Throughout this disclosure, the second channel is also referred to as a bypass or bypass channel. 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 tissues. 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.

[0078] In one embodiment, the microfluidic device may include a single microfluidic module. In such an example, when the restraint is blocked, fluid flows through the second channel and toward the output. In other embodiments, the microfluidic device may include more than one microfluidic module, and thus include more than one trap and restraint within each device. In a multi-module configuration, the second channels of each module may be connected to form consecutive second channels leading to the same output. In some embodiments, the direction of fluid flow through the second channel relative to the object trap is such that the fluid flow is directed toward the second channel once the / each restraint is blocked and the fluid overflows from the object trap into the second channel.

[0079] The multi-module configuration of the microfluidic device with its microelectronic substrate provides a high-throughput device through which electrophysiological activity from multiple tissues can be measured simultaneously to determine the percentage of functional tissues. This feature offers significant advantages, such as when rapid assessment of the quality of a large number of islets is required prior to transplantation. Importantly, the device disclosed herein overcomes the limitations of prior art, which typically requires complex instrument setup, has low throughput, and / or causes damage to the object during manipulation.

[0080] In another embodiment, it is conceivable that the dimensions of the first channel, the object or spherical trap, the restraint, and / or the second channel can be altered to accommodate different tissue types and / or sizes by adding adapters or stops capable of limiting and / or increasing the cross-sectional area. In one embodiment, the microfluidic module includes adapters or stops for varying the cross-sectional area of ​​the first and / or second channels at a defined point within a specified range. In a specific embodiment, channel dimensions may include diameter, height, or width in the range of 200 μm and 500 μm. In an alternative embodiment, the object trap and / or restraint may include adapters or stops to vary the diameter, height, or width of the channels(s) depending on the type of object provided to the microfluidic device.

[0081] Each microfluidic module of this disclosure may include at least one electrode to assess electrophysiological states and / or an object's response to stimulation. The microfluidic device of this disclosure includes at least 10 electrodes, preferably located at each object trap, to enable the detection of the object's electrical activity. A reference electrode is typically required to obtain a background or reference signal of the electric field. In a preferred embodiment, the microfluidic module includes a uniform grounded reference electrode. The end of each electrode extends into the microchannel to enable the acquisition of multiple measurements from within the channel. In one embodiment, each module of the microfluidic device includes at least 10 recording electrodes. In a preferred embodiment, the electrodes of this disclosure include microelectrodes. In some embodiments, the 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 and / or / multiple titanium electrodes.

[0082] However, in configurations including multiple modules, it is not required that all modules include electrodes. In some embodiments, one or more modules of the microfluidic device do not include electrodes. One or more modules lacking electrodes can serve as controls, where the tissue used in the assay can be visualized under a microscope, or for biochemical assays such as gene expression, compound detection, or viability assays using optical detection (e.g., absorbance or fluorescence). In such applications, the one or more modules may include access points through which tissue can be isolated for analysis and through which solutions infused with the tissue can be collected without disturbing the tissue (e.g., for secretion assays). To enable visualization of the one or more tissues via the microfluidic device, in some embodiments, the one or more modules may include polymer coverslips or the bottom of glass coverslips through which objects can be visualized directly via the device. If electrical information is required, transparent electrodes can be printed on these coverslips.

[0083] The microfluidic device can be made of, for example, plastic, glass, silicone, or other materials. In a preferred embodiment, the channel of the microfluidic device comprises transparent polydimethylsiloxane (PDMS). While the examples disclosed herein use transparent PDMS for the channel and borosilicate glass as a substrate for the titanium (Ti) electrodes (which are insulated by a silica deposit), any transparent substrate or molded plastic known in the art suitable for fabricating microfluidic devices can be used.

[0084] One or more electrodes of the device can be fabricated in a variety of ways known in the art. In one embodiment, the microfluidic device according to this disclosure can be fabricated by a printing method, wherein the one or more electrodes are printed in the first channel by, for example, plasma bonding or pressure fixing methods.

[0085] The apparatus disclosed herein may further include a chip holder and / or amplifier connected to computer reading software for reading software. Commercially available amplifiers (such as, but not limited to, the 3600 type amplifier) ​​can be used, which can be used as a multi-channel extracellular differential AC amplifier. For example, the amplifier typically includes at least 16 channels, at least 10 gain settings, at least five low-pass filters ranging from 50 Hz to 50 kHz, at least five low-pass filters ranging from 0.1 Hz to 600 Hz, and a notch filter for each channel (e.g., 50 Hz or 60 Hz). In some cases, the signal may be pre-amplified by a headstage typically located near the electrodes(s), and the recording is collected by the amplifier.

[0086] Those skilled in the art will appreciate that the microfluidic devices of this disclosure can have a variety of applications. In one embodiment, the microfluidic device can be used to assess the electrical activity, survival rate, function, and / or response to stimuli of an object. Stimuli can include any substance that triggers changes in population cellular activity, such as glucose, ions, hormones (multiple), ligands (multiple), electrical stimulation, or drugs (multiple). In a preferred embodiment, the stimulus provided to the microfluidic device includes glucose.

[0087] In a sixth aspect, a method is provided for adding one or more tissues to a microfluidic device as disclosed herein for measuring the electrical activity of the one or more tissues, wherein the method includes: providing one or more tissues in a fluid to an input portion of the device, wherein the fluid initially flows through the device until the one or more tissues reach a trap and obstruct fluid flow through a restrictor. This obstruction of fluid flow by the restrictor facilitates fluid flow through a second channel and to an output portion of the microfluidic device.

[0088] In a seventh aspect, a method for analyzing an object using the microfluidic device of this disclosure is provided, comprising:

[0089] (i) To supply one or more tissues in fluid to the input of the microfluidic device, wherein the fluid initially flows through the device until the one or more tissues reach the trap and block the flow of fluid through the restrictor;

[0090] (ii) Measuring baseline electrophysiological activity of the one or more tissues using the one or more electrodes;

[0091] (iii) Delivering a test solution to the one or more tissues via the input section, the test solution causing the object to become more or less electrically active, the substrate being either a substrate that stimulates electrical activity from specific cells or a substrate that blocks stimulating electrical activity from specific cells, such as for high glucose concentrations in the pancreas; and

[0092] (iv) In response to the test solution, the electrophysiological activity of the one or more tissues is measured through the one or more electrodes.

[0093] In one embodiment, the method may include more than one test solution, wherein steps (iii) and (iv) are repeated, wherein an optional step (v) includes: after each solution, providing a wash solution through an input section to measure the electrophysiological activity of the one or more tissues in response to each test solution.

[0094] In some embodiments, after the tissue is delivered to the trap and the restrictor is blocked, the fluid in step (i) can be removed by aspirating fluid from the output before adding the test solution of step (iii). In an alternative embodiment, the output may initially be blocked by a stop, which is released before adding the test solution of step (iii) to remove the solution in step (i) by gravity and capillary actuation.

[0095] In one embodiment, the one or more tissues (Langehanes islets) are derived from the pancreas. To obtain optimal object survival, one or more tissues, as disclosed herein, are provided to the microfluidic device in solution and / or held in a solution within the microfluidic device. In a preferred embodiment, the one or more tissues are provided to the microfluidic device in solution, such as a commercially available cell culture solution suitable for the tissue type. Once the one or more tissues are provided to the microfluidic device, the tissue(s) travel along the first channel and toward the trap to block fluid flow through the restrictor and to enable measurement of the electrophysiological activity of the object via the electrodes. In one embodiment, the one or more tissues move through the first channel by gravity and capillary-driven fluid flow.

[0096] In one embodiment, the culture solution and / or the test solution may contain glucose. In one embodiment, the glucose concentration 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 glucose concentration in the solution may be any concentration that is physiologically relevant to the condition of interest or tissue type.

[0097] In one application, the microfluidic device and / or method described herein can be used as a drug discovery platform or for the safe screening of compounds. Alternatively or additionally, the culture solution and / or test solution may contain one or more drugs to assess alterations in the electrophysiological responses of (multiple) tissues. The one or more drugs may be therapeutic agents that increase cell viability, and / or agents that alter the electrophysiological responses of the tissues. The one or more drugs may contain ion channel agonists or ion channel antagonists. For example, one or more drugs may contain, for example, tolbutamide, chlorpropamide, or gliclazide. Alternatively, the electrophysiological activity of an object can be manipulated by altering the ion concentration of the extracellular medium. In one embodiment, the test solution contains a higher concentration of a component (e.g., ions, glucose, other nutrients) that comprises the culture solution. Optionally, in some cases, the one or more drugs may contain a hormone that inhibits electrical activity in the object, such as insulin in the case of pancreatic islets.

[0098] The method according to this disclosure enables rapid, accurate, and label-free assessment of tissue viability and / or function. Alternatively, the method may include additional steps of isolating the one or more tissues from the first channel and / or isolating the fluid from the microfluidic device for further analysis. The one or more tissues may be isolated for further analysis, such as assessing object electrical activity, viability, function, and / or response to stimuli. The further analysis may include various techniques known in the art, such as transcriptomic analysis or fluorescence microscopy. In some embodiments, the test solution may contain one or more molecules for labeling the object, wherein the labeled tissue is isolated for further analysis. For example, the one or more molecules for labeling the object may include any commercially available fluorescent probes or dyes for staining cellular components.

[0099] A feature in one aspect can be provided as a feature in another aspect. For example, a method feature can be provided as an apparatus or device feature, and vice versa. Attached Figure Description

[0100] Various aspects of the invention will now be described by way of example only and with reference to the accompanying drawings, in which:

[0101] Figure 1 This is a top view of a multi-channel device microfluidic channel with multiple traps according to an embodiment;

[0102] Figure 2 yes Figure 1 An enlarged view of the device in the image;

[0103] Figure 3 yes Figure 2 Another enlarged view of the device shows an indication of the flow direction and the location where the object will be captured;

[0104] Figure 4 Top and sectional views of another single trap device are depicted as a background example;

[0105] Figure 5(a) is a top view of a single trap device, Figure 5(b) is a view of a multi-trap device according to an embodiment, Figure 5(c) is a photograph of a microfluidic module of a device incorporated into a glass microelectrode array, and Figure 5(d) is an example of a multi-channel microfluidic chip and electrode array mounted in a holding module, and Figure 5(e) is another photograph of a microfluidic module of a device incorporated into a glass microelectrode array.

[0106] Figures 6(a) to 6(d) are additional views and micrographs of the multichannel device, and Figure 6(e) is a composite micrograph showing the pancreatic islets captured in the device.

[0107] Figures 7(a) and 7(b) depict the sensing element module of the device according to the embodiment;

[0108] Figures 8(a) and 8(b) depict the sensing element module of a device according to another embodiment;

[0109] Figure 9 An apparatus according to an embodiment is described;

[0110] Figures 10(a) to 10(d) are graphs of the results derived using this device;

[0111] Figures 11(a) and 11(b) are Figure 1 Another view of the device in the image;

[0112] Figure 12 yes Figure 1 Another view of the device in the image, and

[0113] Figure 13 This is a top view of the device according to an embodiment.

[0114] Figure 14 is a top view of a device with stimulating electrodes according to an embodiment. Detailed Implementation

[0115] This invention relates to an apparatus. According to an embodiment, the apparatus can be used to determine the tissue quality, maturity, or developmental status of Langerhans islets of Langerhans by measuring electrical activity. The apparatus can also be deployed in a similar manner to measure the electrical activity of any other organoids, such as, but not limited to, nerve cells or organoids and neural stem cells of similar size (100 μm to 300 μm in diameter), cardiac spheroids, etc.

[0116] Electrode-based methods for assessing the survival of newly isolated islets are generally considered impractical. For example, in “Functional testing of pancreatic islet cells based on microelectrode arrays” (Alassaf et al., 2020), the use of a planar MEA (microelectrode array) platform was indicated (see [link to study]). Figure 1Extracellular recording of pancreatic islets (Alassaf et al., 2020) using classic MEA chips is not feasible, and recording of electrical activity requires proper contact and adhesion between the islets and electrodes. Given that islets are large, multicellular spheroids, the limited contact area with recording electrodes in planar MEAs hinders MEA recording for functional assessment of the islets. In contrast to the apparatus described herein, the islets of Alassaf et al. were subsequently dissociated into single cells on an array and cultured for several days. Known methods of using imaging-tagged islets render them unsuitable for transplantation purposes. Alternative known methods may damage the islets, again rendering them unsuitable for transplantation. In the following text, the terms organoid, spheroid, islet, and object used as an example of tissue or tissue culture are used. In this sense, an organoid can refer to a cluster of cells derived from embryonic stem cells, induced pluripotent stem cells, or organ-specific adult stem cells that has been developed in culture to form a recognizable structure possessing some or all of the anatomical and physiological / functional properties of a normally developed organ. They typically consist of thousands of cells. These include, but are not limited to, stem cell-derived pancreatic islets, neural stem cell-derived brain organoids, cardiac spheroids, kidneys, ovaries, intestines, and spleens. These tissues or objects have diameters ranging from approximately 100 to 300 micrometers. On the other hand, primary organoids can refer to tissues derived from donor material of the same size, such as donor pancreatic islets, as well as muscle and brain biopsies. The apparatus described below can measure the amount of excitation in excitationable tissues, and the methods for determining function are best applied to excitationable endocrine tissues such as islets, brain, and muscle.

[0117] There is an unmet need in the art for the development of novel devices and methods that can efficiently identify cell viability and functional clusters, including islets for transplantation, without chemical labeling or biochemical manipulation. Rapid identification of islet viability is crucial because transplantation outcomes are typically highly correlated with islet quantity and viability. In the context of islet transplantation as an example, the transplantation process is a multi-step procedure involving pancreas harvesting, tissue dissociation, islet purification, islet culture, and islet transplantation via the portal vein into the recipient's liver. Islet loss occurs at each step (primarily due to ischemia). Furthermore, less than 50% of the islets from a single pancreas (~500,000 islets) are isolated, and of these islets, it is estimated that less than 50% are transplanted into the liver. Therefore, there is an urgent need for novel devices and methods capable of rapidly assessing quality without adversely affecting islet viability and function. This disclosure is based on a method that eliminates the need for complex manipulation of islets (or other objects mentioned, including organoids and cell clusters), requires no chemical manipulation or labeling, and provides the possibility of rapidly testing multiple islet responses (or other objects mentioned) to give a projected estimate of batch survival.

[0118] It will be understood that all living cells possess bioelectrical activity, and generally, many cells can respond to chemical or physical stimuli that can enhance or diminish their bioelectrical activity. For example, cardiomyocytes / cardiomyocyte organoids / stem-derived cardiomyocytes can be stimulated with isoprene, and their electrical activity can be measured. Those cell clusters with viability will generate coherent extracellular electrical signals at rest and in response to such pharmacological or electrical stimulation (pacing), but those clusters without viability will not generate any coherent electrical signals or any electrical signals at all, thus distinguishing between viable and non-viable cell clusters. Therefore, this device can also be applied to stem cell-derived neural objects, other neural cell clusters, and other organoids.

[0119] Figure 1 This is a top view of a device with multiple traps according to an embodiment. It will be understood that the device 100 includes a microfluidic module and an electronic sensing module, and Figure 1Only the microfluidic module is depicted. The electronic sensing module is described in further detail, for example, with reference to Figure 7. The device 100 is configured to capture multiple objects (particularly biological objects), such as tissues and / or multicellular objects. Objects may include cellular objects or tissue objects. In this embodiment, each trap includes a restraint for confining or capturing tissue or organoid-like material in a cell culture. In the following description, the device is described as capturing cellular spheroids; however, it will be understood that the device can be used to capture objects derived from cultures or tissue cultures, such as organoids, spheroids (e.g., object spheroids, tissue spheroids, and islets). Such objects are suspended in a fluid and transported via a fluid flow through channels of the device. The device can be used to capture and test excitable tissues and / or multicellular objects to test bioelectrical activity as a representative of tissue viability.

[0120] The microfluidic module has an input section 110 (also called a common input section or shared input section) and an output section 111 (also called a common output section or shared output section). The microfluidic module also has an additional output section 113. This additional output section 113 is separate from the shared output section 111. The microfluidic arrangement has multiple fluid paths between the input section 110 and the shared output section 111. Figure 1 In one embodiment, 11 fluid paths are defined between the input section 110 and the shared output section 111, with each fluid path passing through a limiting member. As described above, each limiting member is configured to capture one or more tissues and / or multicellular objects in the fluid introduced into the device via the input section 110, and to transport one or more tissues and / or multicellular objects along a corresponding fluid path of the limiting member.

[0121] In addition to the multiple fluid paths defined between the input section 110 and the shared output section 111 via limiting members, an additional fluid path is defined between the input section 110 and an additional fluid output section 113. This additional fluid path is in fluid communication with multiple flow restrictors but does not pass through the limiting members. The additional fluid path has a delivery portion between the input section 110 and the multiple limiting members 108, which allows fluid to be delivered to the limiting members. The additional fluid path has an output portion, which allows fluid to be removed from the device via the additional output section 113. This microfluidic arrangement causes the multiple limiting members to be positioned adjacent to and in fluid communication with the additional fluid path. Therefore, the additional fluid path is arranged to expose the captured and / or restricted object to the additional fluid. This additional fluid path may be referred to as a fluid delivery and / or flushing path. The multiple fluid paths may be referred to as object delivery paths.

[0122] exist Figure 1In one embodiment, the device has a first channel 101 defined between an input portion 110 and a separate output portion 113. The first channel includes an input portion 102 (also referred to as an input channel portion), an intermediate portion 115, and a bypass portion 104 (also referred to as a bypass channel portion). The input portion 102 is connected to the intermediate portion 115 at a first connector 113, and the intermediate portion 115 is connected to the bypass portion 104 at a second connector 112, such that the intermediate portion 115 is disposed between the first connector 113 and the second connector 112. The device has a second channel 106 (also referred to as an outlet channel) connected to the first channel 101 via limiting members. The second channel has a first portion 106a and a second portion 106b. The second channel 106 is connected to the first channel along the first portion 106a via a plurality of limiting members 108. Specifically, a plurality of limiting members 108 arranged along the middle portion 115 of the first channel connect the middle portion 115 to the first portion 106a of the second channel 106. The input portion 102 and the middle portion 115 can together form a fluid delivery portion and / or an object delivery portion. The bypass portion 104 can form a fluid flushing portion and / or an object flushing portion. Therefore, a plurality of limiting members can be provided at or on the fluid delivery portion and / or the object delivery portion.

[0123] What will be understood is... Figure 1 The dimensions of the first and second channels in the device are designed to allow fluid flow of the object. Therefore, in this embodiment, the first channel is sized to allow flow of the object of interest, and the limiting element is sized to prevent flow of the object of interest. In this embodiment, the object of interest is a cellular spheroid. Typically, the size of the object of interest can range from 150 micrometers to 300 micrometers. In this embodiment, the first and second channels have a width of 450 micrometers and a height of 250 micrometers, and the limiting element is 40 micrometers. It will be understood that alternative dimensions can be used in alternative embodiments. In particular, these design parameters can be selected according to the object of interest. The channels are formed in a PDMS layer with dimensions of 3cm × 3cm × 0.5cm.

[0124] exist Figure 1 In one embodiment, an additional fluid path is defined by a first channel 101 between the input section 110 and the additional output section 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 section 110 and the output section 111. Specifically, in Figure 1In one embodiment, multiple fluid paths are defined between the input section 110 and the output section 111 via the input channel portion 102, the middle portion 115 of the first channel, the first portion 106a of the second channel, and the second portion 106b of the second channel.

[0125] exist Figure 1 In this embodiment, the delivery portion of the additional fluid path is located between the input 110 and the connection point 112, and 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 plurality of fluid paths between the input 110 and the connection point 112. It will be understood that the bypass portion 104 of the additional fluid path is spatially separated from the plurality of fluid paths. Therefore, the additional fluid path provides a route for flushing and / or removing objects and / or debris from the device via the additional output 113.

[0126] What will be understood is that, Figure 1 In one embodiment, the input channel portion 102 is a common or shared input channel portion used to deliver fluid to each of a plurality of traps. Figure 1 It is also clear that the bypass channel section 104 is a common output channel section for multiple traps, and the outlet channel provides a common output channel for multiple traps. Figure 1 In one embodiment, device 100 has 11 traps; however, it will be understood that in other embodiments, a different number of traps may be provided.

[0127] Between the fluid inlet 110 and the second connector 112, the first channel 102 has an input channel portion 102 and an intermediate portion 115. The input portion can be an inlet port, and / or the output portion can be an outlet port. A plurality of traps 108 are specifically arranged along the intermediate portion 105 between the input and output portions of the first channel 102. Figure 1 As shown, each of the plurality of traps 108 is in fluid communication with the first channel portion 102, the bypass channel portion 104, and the outlet channel 106. Figure 1 In one embodiment, multiple traps are provided in series, such that in use, fluid flowing through the input channel 102 is supplied to each cell spheroid in the cell spheroid and then captured, and the multiple traps have a common input channel.

[0128] The fluid flow direction can be defined starting from the input section 110. In the fluid flow direction between the input section and the additional output section 113, it will be understood that the output section of the additional fluid path is downstream of the conveying section and the multiple limiting members.

[0129] exist Figure 1 In the above embodiments, the input portion 111 corresponds to the inlet port, and the output portion corresponds to the outlet of the device. It will be understood that in some embodiments, fluid paths may be defined between input and output portions that do not correspond to inlets / outlets. For example, multiple fluid paths may be defined between an input portion and / or a shared output portion, the input portion being defined at a point along the input channel 102 (e.g., at a point substantially at the connector 113), and the shared output portion being defined along the output channel 106. Similarly, another fluid path may be defined between the same input portion and another output portion, the same input portion being defined along the input channel 102, and the other output portion being defined along the bypass channel portion 104. In such embodiments, more than one inlet may be connected to the defined input portion, and / or more than one outlet may be connected to the shared output portion and / or the other output portion. In such embodiments, the additional fluid path has a delivery portion and an output portion, the delivery portion overlapping with multiple fluid paths, and the output portion spatially separated from multiple fluid paths.

[0130] As mentioned above, in Figure 1 In this embodiment, the channel defines a fluid path through the device. Specifically, for each trap, there is a corresponding fluid path from the fluid inlet 110 to the shared output 111, and the limiting member is a limiting member for that corresponding fluid path. Therefore, multiple fluid paths are defined between the fluid inlet 110 and the shared output 111 via multiple limiting members. Furthermore, in Figure 2 In this embodiment, as described above, the first channel 101 defines an additional fluid path through the device, which is a means of bypassing the restrictors. For each trap, the fluid path delivers the object to the restrictor of the trap. Thus, the restrictor prevents the object from flowing along the fluid path. The additional fluid path is in fluid communication with each of the restrictors and can be used to deliver additional fluid to the trapped object to allow for fluid exchange and / or more fluid delivery. Each of the fluid paths is defined between the input section 110 and the shared output section 111 via its respective trap. Thus, each of the fluid paths at least partially overlaps with the additional fluid path between the input section 110 and the bypass channel section 104; in particular, the overlapping portion corresponds to at least a portion of the intermediate section 115 and the input section 102.

[0131] Figure 1 A more detailed description Figure 2 Two of the multiple traps in the process. Figure 2 The first trap 108a and the second trap 108b are depicted. (See image.) Figure 4As shown, the first trap 108a has a limiting member 114a. The second trap 108b also has a limiting member 114b. It will be understood that each of the plurality of traps in the device 100 has a corresponding limiting member. As described below, the limiting member of each trap is substantially as shown in the reference. Figure 2 Operate as described above.

[0132] More specifically, the first channel 101 (particularly 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 1 The input portion 120a, output or bypass portion 122a, and feeder channel portion 116a of a first trap 108a are depicted, as are the input portion 120b, output or bypass portion 122b, and feeder channel portion 116b of a second trap 108b. These traps are provided in a series arrangement, such that the bypass portion of the trap is connected to the input portion of a subsequent trap in the series. In this embodiment, the bypass portion 122a of the first trap is connected to the input portion 120a of the second trap. Figure 2 , Figure 3 and Figure 4 In one embodiment, for each trap, the input portion (120a, 120b) is substantially perpendicular to the corresponding bypass portion (122a, 122b). In other embodiments, the input portion is configured at an alternative angle to the bypass portion.

[0133] The feeder channel portions (116a, 116b) are sized to receive and hold objects and can function as objects including chambers. Each trap is arranged such that an outlet channel 106 is in fluid communication with the feeder channel portions (116a, 116b) via its respective limiting members (114a, 144b). Each trap can be considered to have an outlet channel portion that forms part of an outlet channel 106, such that limiting members are disposed between the feeder channel portion and the outlet channel portion.

[0134] It will be understood that each limiting element is formed in a fluid channel defined by a portion of the feeder channel 116a and a portion of the outlet channel 106. See reference... Figure 3 As described, each trap can be in a first configuration (referred to as the open configuration) and a second configuration (referred to as the closed configuration).

[0135] In this open configuration, the first channel 101 is in fluid communication with the outlet channel 106 via a restrictor. For the first trap, in this open configuration, the fluid path is limited between the inlet channel 101 and the outlet channel 106 via a restrictor 114a. In this configuration, fluid is thus allowed to flow from the feeder channel portion 116a via the restrictor 116a to the outlet channel 106.

[0136] In this closed configuration, the first channel 101 is not in effective fluid communication with the outlet channel 106 due to a blockage and / or obstruction at the restrictor 114a. In this embodiment, the blockage is formed by a tissue sphere held in the feeder channel portion 116a. The tissue sphere is embedded or otherwise seeded in the chamber. In particular, in this embodiment, the tissue sphere is substantially fixed in the chamber. It will be understood that the tissue sphere is permanently or at least fixed for a sufficient period of time to allow a signal to be sensed by the electrodes. Figure 3 Cellular spheroids 120 are depicted being fixed or captured in the first trap 108a. It will be understood that, with reference to... Figure 4 The described cellular spheroids are merely examples, and in other embodiments, the captured object may be at least one of the following: organ, tissue spheroids, and / or pancreatic islets, cells, or tissue objects. In some embodiments, the object may be an islet extending from the pancreas. Because the size (diameter or width) of the cellular spheroid is larger than the opening of the limiting member 114a, the cellular spheroid forms a barrier to fluid flow, thereby preventing substantially all fluid in the first channel from flowing from the first channel 101 to the outlet channel 106. (See reference...) Figure 3 The trap can be moved from a first configuration to a second configuration by performing a cell spheroid seeding process using the device. When the trap is in the closed configuration, fluid bypasses the trap and flows to subsequent traps in series (via the bypass portion of the closed trap and the input portion of the subsequent traps).

[0137] The plurality of traps are configured such that one or more traps can be blocked by cellular spheroids. When one trap is blocked, fluid flows substantially through a fluid path defined between inlet channel 101 and outlet channel 106, and through any of the unblocked traps. If all traps are blocked, fluid bypasses all traps and flows substantially through an additional fluid path defined along inlet channel 101, defined between inlet channel portion 102 and bypass channel portion 104.

[0138] Figure 3A first trap 108a in a closed configuration and a second trap 108b in an open configuration are depicted. Figure 3 As shown, the first trap 108a has a unit spherical body 120 within a unit spherical chamber, which forms a barrier to fluid flow from a first side of the restrictor (unit spherical chamber side) to a second side of the restrictor (second channel 106 side). In this closed configuration, fluid flows along the input channel 106 bypassing the first trap 108a and then enters the second trap 108b. More specifically, the fluid bypasses the restrictor and exits the first trap 108a via a bypass portion 122a, and enters the second trap 108b via the input portion 120b. In some embodiments, the fluid flow through the trap is shut off or at least reduced in the closed configuration compared to the open configuration.

[0139] like Figure 4 As shown, the second trap 108b is in an open configuration, thereby allowing fluid to flow from the first side (spherical chamber side) of the restrictor to the second side (second channel 106 side) of the restrictor. In this open configuration, fluid flows along the inlet channel 101, and at least some of the fluid passes through the restrictor 114b to reach the outlet channel 106.

[0140] In the above embodiments, a restraining element is described. This restraining element cooperates with the chamber to capture tissue, and therefore, the restraining element can be considered to operate together as a trapping element configured to capture cellular spheroids. It will be understood that alternative restraining elements and / or trapping elements may be provided in alternative embodiments. For example, a mesh or grid structure may be provided to restrain cells and / or other objects or tissue cultures while allowing fluid flow. Reference Figure 1 Additional examples of capture elements are provided.

[0141] In use, multiple objects in the fluid are introduced into the microfluidic device via the input section 110. The fluid initially flows through the device along multiple fluid paths via the input section 102 and the restraints 108. As described above, the multiple objects in the fluid then reach the multiple restraints and are captured and / or at least restrained by the restraints. Once an object is captured at the corresponding restraint, a sensing process is performed to obtain sensor signals of the multiple captured objects using multiple electrodes. In some embodiments, the fluid flow is achieved through gravity and / or capillary action.

[0142] The sensing process can be performed before or after additional fluid is delivered to the captured objects. Additional fluid is introduced into the device via input 110 and supplied to the captured objects via an additional fluid path. The fluid then flows to an additional output 113. The sensing process includes sensing sensor signals from electrodes disposed at each trap. In this embodiment, the sensor signals represent or at least indicate the biological activity of multiple captured objects (objects in this embodiment). A first sensor signal (representing or indicating baseline electrophysiological activity for multiple objects) can be sensed before additional fluid is delivered, and further signals (representing or indicating the electrophysiological activity of multiple objects) are sensed in response to the fluid.

[0143] What will be understood is that, although Figure 4 Multichannel microfluidic modules are depicted, but more than one of these modules (e.g., as an array) can be provided to offer additional channels. Such combinations of modules can be accompanied by corresponding arrays or appropriately larger arrays of microelectrodes, as described below.

[0144] Figure 4 (The above figure) depicts a fluid device having a first channel 12 and a second channel 20. It describes... Figure 4 This illustration shows the operation of a single trap for background information. A first channel 12 has a fluid inlet 14 and a fluid outlet 16, and a trap is provided between the inlet 14 and the outlet 16. The trap has a limiting member 18 disposed in the first channel 12 between the inlet 14 and the outlet 16. The limiting member 18 can be considered to define a feeder channel portion 12a and an outlet channel portion 12b of the first channel. The feeder channel portion 12a can also be referred to as a feeder channel, and the outlet channel portion 12b can also be referred to as an outlet channel. The limiting member 18 is sized to allow fluid flow between the feeder channel portion 12a and the outlet channel portion 12b, and the limiting member 18 is sized to prevent one or more cellular spheroids from passing through the limiting member. The limiting member can be considered to be an opening formed by two members 19a, 19b disposed in the first channel. A second channel 20 is in fluid communication with the feeder channel portion 12a, and the second channel 20 can also be referred to as a bypass channel. The trap also has at least one unrelated reference electrode or ground electrode, and a measuring electrode (c') for detecting electrophysiological signals, such as those from one or more cellular spheres trapped in the trap. The use of the trap to detect electrophysiological signals is described elsewhere.

[0145] It will be understood that the channels define the fluid path through the device. Specifically, a first fluid path exists from the input 14 to the output 16, and the limiting member 18 is a limiting member on this fluid path. Additionally, a further fluid path is defined between the input 14 and the second (or bypass) channel 20. This first fluid path uses fluid flow to transport the cell spheroids to the limiting member of the trap. Thus, the limiting member prevents the cell spheroids from flowing along the first fluid path. The further fluid path is in fluid communication with the limiting member and can be used to transport additional fluid to the trapped cell spheroids to allow fluid exchange, and / or to transport additional fluid to the trapped cell spheroids. The first fluid path and the further fluid path at least partially overlap.

[0146] In use, the trap can be in one of two configurations: an open configuration and a closed configuration. In the open configuration, the feeder channel portion 12a is in fluid communication with the outlet channel portion 12b via the restrictor 18. In this open configuration, fluid is allowed to flow along the first channel from the feeder channel portion 14 via the restrictor 18 to the outlet channel portion 16.

[0147] In the closed configuration, the feeder channel portion 12a is not in fluid communication with the outlet channel portion 12b due to blockage and / or clogging at the restrictor. In use, this blockage is formed by cell spheroids held in the feeder channel portion 12a at the restrictor 18. Because the size (diameter or width) of the cell spheroids is larger than the opening of the restrictor, the cell spheroids, together with the components 19a, 19b in the channel 12, form a barrier to fluid flow, preventing substantially all fluid in the first channel from flowing from the feeder channel portion 12a to the outlet channel portion 12b. Therefore, when the restrictor is blocked, fluid flows to the bypass channel 14 at a faster rate. By using this device to perform the cell spheroid seeding process, the trap can be moved from the first configuration to a second configuration. When in the closed configuration, it will be understood that fluid flows to the bypass channel at a faster flow rate than when in the open configuration.

[0148] As described above, the device is a combination of a transparent microfluidic channel design and a "trapper," which is provided together with the printed microelectrode array. Figure 4 A single-channel example is given. Figure 4 In the embodiment, the channel dimensions in the feeder channel and the outlet channel are 450 μm wide and 250 μm deep (see [reference]). Figure 4The confinement in both the main and bypass channels of the trap is 20 μm to 50 μm (a value selected based on the required flow rate). The width of the confinement (i.e., the narrower channel) can be selected from a suitable range to ensure uniform flow in both multi-channel and single-channel systems. In some embodiments, this width includes a range of 20 μm to 50 μm. Waste exits the system via an output channel, which can also be sampled to assess the secretion of insulin or other substances (see Figure 5).

[0149] Figure 5a Additional details are provided regarding the arrangement and dimensions of the channel microfluidic and electrode array (showing a single electrode as part of a single-channel example). a. Input channel, 450 μm in size; b. Constraint, 40 μm wide; c. Reference electrode; c'. Measurement electrode; d. Profile at cross-section; e. PDMS channel cross-section; f. Silicon oxide insulator (100 nm thick - not drawn to scale); g. Gold electrode tracks and electrode pads; h. Glass substrate; i. Channel height (250 μm); j. Channel width (450 μm as in a).

[0150] Figure 5 provides further details of the channel layout as a simplified example. Figure 4 .exist Figure 5b The diagram shows the arrangement of the input and output channels of a single microfluidic chip, with bypass arrows indicating the flow direction. Figure 5c .Diagram showing the channel bypass of a multi-channel microfluidic device. Arrows indicate the flow direction (the channel dimensions are the same as those of a single-channel device). Figure 5d Photograph of the assembled multichannel electronic chip, with microfluidic modules attached. Figure 5e Multichannel microfluidic chip and electrodes installed in the holding module. Figure 6a Additional photos of the microfluidic chip and electrode array.

[0151] Figure 6 provides a detailed drawing of the glass substrate with a printed electrode array and demonstrates a functional example of a multi-channel device. Figure 6b An overview of the alignment of electrodes and microfluidic circuits. Figure 6c Details of the electrodes and microfluidic traps, e' is the electrode track, f' is the reference electrode, and g' is the microfluidic channel. Figure 6a .like Figure 6d The channel in the image shows the capture of a 150 μm glass sphere (h'). Figure 6e A linear channel arrangement was shown, which demonstrated the capture of microfluidics on a 150 μm glass sphere on the electrode. Figure 4 Composite micrographs showing instances of pancreatic islets captured on an assembled microfluidic module and microelectrode array.

[0152] In some embodiments, single or multiple microfluidic channels use capillary action to deliver islets to a “trapper” without the need to apply negative or positive pressure (e.g., see [link to documentation]). Figure 6e Figure 5 and Figure 4 Once the islets are captured, the main channel is effectively blocked, and the microfluidic bypass allows the test solution to flow through (see, for example, [link to relevant documentation]). Figure 6e and Figure 10a ).

[0153] The “flow” allows for the rapid exchange of the normal extracellular solution (also via capillary action and / or gravity) with solutions containing higher concentrations of glucose (e.g., 15.5 mM glucose), washes, drugs, or dye-labeled solutions for object counting or evaluation. Due to glucose sensing, the increased glucose concentration induces an increase in the electrical activity of pancreatic β-cells in the object (see [link to relevant documentation]). Figure 10b and Figure 4 Since β cells are the majority of glucose-sensing cells in the islets, measurable electrical changes during activity can be detected via 30 μm electrode pads, with the islets fixed on top of the electrode pads relative to a larger “ground” electrode (see [link to electrode description]). Figure 9 This electrical response is observed only in healthy, fully functional islets. The chip's electrical output is "read" by connecting the array to larger printed metal contact pads via electrical contacts connected to electrode pads located beneath each islet via insulated tracts. Processing of the electrical signals produces an indication of whether the islets are responding.

[0154] The device is designed to provide a timely assessment of batch islet survival. A prototype has been built and has been used to assess islet quality (see [link]). Figure 10a D、 Figure 10b and Figures 1 to 3 It is anticipated that approximately 30 minutes will be spent evaluating a batch of islets, with the challenge being to first use a lower concentration of glucose, followed by a higher concentration. These results will then be compared with other survival assays.

[0155] It is conceivable that islet samples from donor pancreas could be pipetted into such a device, and microfluidic channels and traps would be used to separate and immobilize the islet samples onto electrodes. The addition of glucose and monitoring of electrode activity would be used to provide a survival percentage using RMS (root mean square noise) measurements. This process would take a maximum of 2 to 3 hours. Currently, in human islet laboratories, islets are cultured for up to 48 hours, and therefore, this timeline would fall well within that margin.

[0156] It will be understood that this device is a hybrid of an electronic device and a microfluidic device. Figure 5(c) is an image of the device in use according to an embodiment. The device has a microfluidic module 52 and an electronic module 54. A means for supplying fluid to the microfluidic module (a pipette 56 in this embodiment) is also shown. The microfluidic module has several limiting elements, substantially as referenced above. Figure 13 As stated above.

[0157] Figure 5(c) is a first photograph of the device, 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 is optionally referred to together with the electronic module as a microfluidic chip. A holder 56 is also shown in Figure 5(d). The holder (also called a chip holder) is sized and shaped to provide a support for the chip. In particular, the holder is essentially U-shaped. This gap allows for inspection of the chip while it is held in the holder. The gap has edges to provide support for the device, particularly for the microelectronic module. When the microelectronic module is held by the holder, the microelectronic module is positioned on the edge of the holder, and the microfluidic module is positioned on top of the microelectronic module.

[0158] Figure 5(e) also depicts input port 58, first output port 60, and second output port 62, which correspond to reference... Figure 9 The description includes an input port 1308, a first output port 1302, and a second output port 1304. In this embodiment, the input port 60 (also referred to as the inlet) has a diameter of 1 mm, and the output port 62 (also referred to as the flush outlet) has a diameter of 1 mm. The second output port 64 (also referred to as the waste outlet) has a diameter of 6.0 mm. The inlet and outlet are formed by creating voids or cutouts in the PDMS layer.

[0159] Figures 7(a) and 7(b) depict schematic diagrams of the electrode configuration of the electronic module. A glass electrode array for a 15-channel device according to an embodiment is depicted. The sensing module is disposed on a glass substrate layer 702 with dimensions of 5 cm x 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) depicts an enlarged view of the electrode configuration 704. A plurality of electrodes (labeled 1 to 15 in this embodiment) are provided such that the corresponding endpoints of the plurality of electrodes are linearly arranged and the corresponding endpoints define a straight line. The end of the first electrode 708 is indicated in Figure 7(b). A single reference electrode 706 spans the width of the plurality of electrodes. The electrode configuration is operable to sense a signal relative to the reference electrode for each electrode. When used with a microfluidic module, each electrode is aligned at or at a corresponding constraint to allow sensing of a signal of a captured object. Figure 6(e) above shows the captured islets of Langerhans and the electrodes.

[0160] It will be understood that the device can have different numbers of channels and constraints. Figure 8(a) depicts the electrode configuration of a 60-channel device. Figure 8(a) depicts 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). It will be understood that in the embodiment of Figure 8(a), four 15-channel microfluidic modules are combined for use with the electrode configuration. The sensing modules in Figure 8 are mounted on a 10cm × 5cm glass substrate. Figure 8(b) depicts two 15-electrode modules together forming part of a 30-channel device. In Figures 8(a) and 8(b), the modules are arranged such that the reference electrodes in each module are parallel. In Figures 8(a) and 8(b), the modules are arranged such that the linear arrangement of the electrode ends is parallel.

[0161] It will be understood that a sensor signal can be sensed between the reference electrode and each trap electrode. In some embodiments, a sensor signal is sensed for each trap during the sensing process. In some embodiments, signals from each trap / channel are isolated from each other. The reference electrode may be a ground reference electrode. The ground electrode is positioned such that it overlaps with at least a portion of another fluid path and / or multiple fluid paths.

[0162] In the above embodiments, a hybrid microfluidic device and electronic device are described. At least a portion of the device can be configured as a "cartridge" for use with other devices. Figure 9 A device 900 according to one embodiment is depicted. Figure 9 In this embodiment, processing resources are provided as part of a larger device configured to receive a removable chip. This removable cartridge or removable chip may correspond to the device described above. In some embodiments, it is a removable cartridge.

[0163] Device 900 includes one or more displays 902, a chip holder 904 with a housing 904, and provided processing resources, in this embodiment, provided as part of a data processing PC 908. The device is configured to receive a disposable chip 906 via a first cylinder opening 908 in a first surface (the upper surface of the housing 904). One or more fluid openings may be provided in the upper surface of the housing. In this embodiment, the fluid openings are sized to receive a pipette. In this embodiment, the microfluidic module of the cylinder includes 60 channels (in four separate groups, each group having a separate fluid inlet). Therefore, a manifold is provided, either as part of the device or as a removable portion for use with a pipette, to direct received fluid to each separate fluid inlet. In some embodiments, the fluid inlets and cylinder openings are aligned such that when the inlet of the microfluidic module is inserted into the cylinder opening, the opening of the microfluidic module aligns with the fluid opening to allow fluid delivery to the inserted cylinder. The processing resources can be configured to run software that performs automated or at least partially automated data analysis on the sensing signals from the cylinder.

[0164] Additional devices may also include user input devices (not shown) to allow users to interact with and control the device. The user input devices may allow users to control the sensing process, and / or data analysis, and / or select different results to view. As a non-limiting example, in some embodiments, the device may be operable to allow users to select the number of channels to be used (e.g., up to 60) via the user input devices.

[0165] The device may also include a camera or other suitable imaging device to allow image capture of the tube during use. The device may be operable to allow, for example, selection of different captured images during data acquisition. This selection can be made using a user input device. The device is also operable to allow the user to run tests on a subset of the available captures. For example, the user can determine that only a subset of the captures has existing islets and therefore select to run tests only on those channels.

[0166] The display can also be configured to show results. In this embodiment, the display shows the percentage of surviving islets. In some embodiments, the device can be operable to allow a user to select a result to be displayed using a user input device. The user input device can be any suitable user input device (such as a mouse or keyboard). In some embodiments, the display forms part of the user input device.

[0167] In some embodiments, at least a portion of the "tube" is disposable. For example, the microfluidic portion may be disposable.

[0168] In some embodiments, the removable cartridge comprises only the microfluidic portion of the device, and the electronic module is provided separately (e.g., as part of the device, or as an additional component to be combined with the cartridge).

[0169] Not in Figure 10a The diagram shows additional computing and / or storage resources located inside the housing. For example, it will be understood that in some embodiments, the device has a memory storage device for storing data, and / or the device can be connected to a network to allow data to be transferred from the device to another computing device. In some embodiments, the device may have a removable storage medium. An amplifier may also be provided for amplifying one or more signals sensed by the electrodes.

[0170] The processing resources are configured to process signals from the electrodes or data from the signals. Processing of the signals and / or data may include determining the percentage of surviving objects (e.g., tissues and / or cells) in the sample. Alternatively or additionally, processing of the data may include determining at least one of electrical activity, survival rate, function, and / or response to stimuli, and the processing may include generating data representing electrical activity, function, and / or response to stimuli, and storing the data.

[0171] In some embodiments, the processor is configured to determine a measurement of the electrical activity of the captured object in response to an additional fluid (an additional fluid introduced via an additional fluid path). The processor may be configured to determine the number of surviving objects and / or functional tissue objects and / or multicellular objects in the sample, or to perform diagnostic and / or survival indications based on electrical activity sensed at multiple restraints and / or sensing areas, for example, where the diagnostic and / or survival indications may be based on electrical activity in response to exposing the object to an additional substance, which may optionally comprise chemicals and / or drugs (e.g., glucose).

[0172] While the example of glucose has been described, it will be understood that other types of additional fluids may be used. In particular, a washing solution may be introduced to remove any solution from the device. Alternatively, molecules for labeling the captured object may be introduced, or one or more drug stimulants or inhibitors may be introduced and / or one or more cellular activity stimulants or inhibitors may be introduced.

[0173] Figure 10 depicts the results of electrical measurements from human pancreatic islets. Figure 10bPancreatic islets exposed to 3 mM glucose followed by exposure to 15 mM glucose; glucose-stimulated insulin secretion (GSIS) measurements show the electrical response at the upper trace with 15 mM glucose and the RMS (root mean square) of electrical noise at the lower trace with 15 mM glucose. It is noted that there was no insulin secretion and no corresponding bioelectrical activity when using 3 mM glucose. Figure 10c GSIS stimulation from 5.5 mM glucose to 15.5 mM glucose showed results. And... Figure 10d The results of RMS measurements are shown to illustrate the sensitivity of the GSIS response at 7.3 mM glucose followed by 17.3 mM glucose (the results were significantly different between the two glucose concentrations, where the glucose concentration was at the student paired test T at the P < 0.0001 level, N = 11). Figures 1 to 3 The percentage of islet survival was measured, in this case, in three batches of islets examined following responses to 3 mM glucose and then to 15 mM glucose; the number of islets in each batch showing electrical activity in response to 15 mM glucose was calculated and expressed as a percentage of islets isolated from the three batches (N=16, N=13, and N=16 in each batch of islets tested). It is noted that the 3 mM glucose solution served as a control condition, in which insulin secretion or electrical activity of the islets was not expected.

[0174] Figure 11(a) depicts a device including a microfluidic module and a sensing element module, the microfluidic module being used as a reference. Figure 2 The microfluidic module described. Figure 11(c) depicts... Figure 12 The device and the captured object, in this case, are spherical. Figure 1 A cross-sectional view of the apparatus is depicted along the line X-X' marked in Figure 11(a). It will be understood that the apparatus in Figure 11 has the same characteristics as the reference... Figure 12 The described apparatus has the same features; however, for clarity, several reference numerals have been omitted.

[0175] Figure 11(a) depicts a first electrode 1102, a second electrode 1104, and a reference electrode 1106. It will be understood that the first electrode 1102 is aligned with the first trap 108a, and the second electrode 1104 is aligned with the second trap 108b. Specifically, the first electrode 1102 is aligned along the opening of the first restrictor of the first trap 108a, and the second electrode is aligned along the opening of the second restrictor of the second trap 108b.

[0176] 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 depicted in FIG. 11(a). The elongated portion is parallel to the opening of the constraint, and in particular, when viewed from above, the elongated portion appears to pass through the opening of the constraint. Therefore, if the constraint opening is centered on the constraint axis and the elongated portion is aligned with the electrode axis, the constraint axis and the electrode axis are arranged in parallel. It will be understood that the second electrode is aligned with the second constraint of the corresponding second trap; however, it will be understood that one or more electrodes may not be aligned with the constraint, for example, the first electrode and the 15th electrode in FIG. 7(b). In some embodiments, the elongated portion may be an electrode track, and the end may be referred to as an electrode pad. In some embodiments, the elongated portion corresponds to an electrode trace, and the electrode end is the electrode itself or at least includes a sensing portion of the electrode. It will be understood that, as referenced... Figure 12 As described, in use, the elongated portion is insulated by an insulating layer, and the electrode tip comes into contact with the fluid and / or the captured object itself.

[0177] The electrodes are arranged such that the end portion of each electrode is substantially positioned in the region corresponding to the trap chamber. The ground electrode 1106 is positioned substantially along the additional fluid path and the delivery portion of the multiple fluid paths. Therefore, a distance is provided between each electrode end and the reference electrode. Figure 11(b) depicts the apparatus of Figure 11(a), wherein two captured objects 1110a, 1110b are in the captured position.

[0178] Figure 11a Depicting in Figure 12 A cross-sectional view of the electrode arrangement in the trap at cross-section X-X'. Figure 12 As can be observed, the device has a PDMS layer 1202, an insulating layer 1204 (in this embodiment, the insulating layer 1204 is a silicon oxide insulating layer), 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, multiple channels are formed in the PDMS layer to form a microfluidic module. Figure 11b A chamber 1208 of the trap is also depicted, which is defined within a PDMS layer by the channel. The chamber has a height and a width. The channel forming the chamber has a height of 250 μm and a width of 450 μm.

[0179] Figure 12 The positions of the electrodes relative to the trap and relative to the captured organoid 1110b are also depicted. The electrodes are disposed on a glass substrate, and a portion of the electrodes is insulated by an insulating layer.Figure 1 A first electrode 1102 on a glass substrate 1206 is depicted, the first electrode 1102 having an elongated portion 1102a and an electrode tip 1102b, as shown with reference to FIG11(a). The electrode is aligned below the chamber such that the electrode tip is disposed in the central portion. In this embodiment, the non-insulated portion of the electrode (electrode tip) is exposed to the channel above. In use, in this embodiment, the non-insulated portion of the electrode contacts a captured object in the trap. In some embodiments, the non-insulated portion of the electrode is adjacent to the captured object and in contact with a fluid comprising the captured object.

[0180] It will be understood that microchannel modules (e.g., reference) Figure 13 The described module is formed in the PDMS layer. Figure 1 Modules according to an embodiment are depicted. (Except for reference...) Figure 13 In addition to the described features, Figure 1 The module also has an input port 1308, a first output port 1302, and a second output port 1304. (See reference...) Figure 1 The inlet port is an inlet located at the fluid input section. (See reference...) Figure 1 The first output port is an outlet located at the shared output section and can be referred to as the waste output section. (See reference...) Figure 13 The second output port is an outlet located at the first output section and may be referred to as a flushing output section. In this embodiment, both the inlet and the outlet are circular. Figure 1 The channels and ports are formed in the PDMS layer 1310, which has a width and length of 3cm and a height of 0.5cm.

[0181] Figures 14(a) and 14(b) depict an electrode array and associated microfluidic module according to another embodiment. The device in Figure 14 adds a stimulation electrode that, when connected to a ground electrode circuit, is operable to provide electrical stimulation pulses across the captured object.

[0182] As can be seen from Figure 14(a), the electrode array is essentially as described with reference to Figure 7, but with the following differences. As described with reference to Figure 7, a plurality of electrodes 1702 are provided such that their respective endpoints are arranged linearly. This endpoint can form part of its sensing element. In this embodiment, the center electrode (labeled 1404) of the plurality of electrodes is a reference electrode. This reference electrode is a ground electrode. In this embodiment, a single electrode 1406 spans the width of the plurality of electrodes and is a stimulation electrode. As can be seen from Figure 14, the center electrode 1404 is wider than the other electrodes of the plurality of electrodes. In the embodiment of Figure 14, the reference electrode and the stimulation electrode form a stimulation electrode pair, and the sensing electrode is configured to sense a response from a stimulation signal (which is a stimulation signal generated by the stimulation electrode pair).

[0183] As described with reference to the embodiments above, the electrode array is aligned with the microfluidic device 1408, which is substantially as described with reference to, for example... I. Qualitative islet responses The description differs from the previous one. Similar to the microfluidic device described above, the second channel of the microfluidic device in FIG. 14 has a first portion 1416a and a second portion 1416b. In this embodiment, the second portion 1416b is centrally located within the first portion while the second channel 106 is connected to the first channel via a plurality of limiting members along the first portion 1416a. The bypass portion and the delivery portion maintain the same configuration. The central reference electrode is aligned such that its length is parallel to the centrally located second portion 1416b of the microfluidic device. In this embodiment, a plurality of fluid paths are defined between the input portion (1422) and the output portion (1424) via limiting members, wherein the fluid paths include a portion of the first portion 1416a and the second portion 1416b. The limiting members are aligned along an axis, and the stimulation electrode is arranged parallel to the axis. The stimulation electrode overlaps with the plurality of fluid paths.

[0184] The electrode configuration is operable to sense a signal relative to a reference electrode for each electrode. Compared to the embodiments described above (where each electrode was aligned with or adjacent to a corresponding constraint to allow the signal of the captured object to be passively sensed), the embodiment of FIG14, in particular, has a single electrode 1406 configured to generate a stimulating electrical signal. Therefore, the single electrode 1406 can be referred to as a stimulating electrode. The plurality of electrodes 1402 can then be referred to as sensing electrodes or recording electrodes.

[0185] Electrical stimulation can directly induce simultaneous action potential discharges in an object, which can be measured and processed by recording electrodes. As an example, direct stimulation can be used for "pacing" of the spherical anterior and other excitable tissues.

[0186] By aligning the electrode array with the limiting element of the microfluidic device, it becomes possible to deliver a high current density to the object.

[0187] Figure 14(b) depicts an enlarged view of the array and microfluidic device in Figure 14(a). It can be seen that at each chamber / sensing region / restriction, electrodes are arranged to form a certain distance between the stimulating electrode 1402 and each of the plurality of sensing electrodes. In this embodiment, a distance 1410 is depicted between the endpoint of the sensing electrode (e.g., 1402i) and the nearest portion of the stimulating electrode 1406. Due to the arrangement of the electrodes, 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 due to the contents of the chamber, the corresponding sensing electrode senses an electrical response across this distance. This electrical response can be a measured voltage. A central reference electrode measures a reference voltage, which can be used as a reference value for each measurement.

[0188] In this embodiment, a single stimulation electrode is described as being arranged perpendicular to the plurality of sensing elements. In other embodiments, different electrode arrangements may be used. For example, more than one stimulation electrode may be provided, and / or a pair of stimulation electrodes and a pair of sensing electrodes may be provided.

[0189] A non-limiting method using the apparatus in Figure 14 is described below. First, as described above, one or more objects are captured. The captured objects in each chamber are positioned on passive electrodes (or in the sensing region of the chamber surrounding the passive region). The passive electrodes are configured to detect electrical activity in the sensing region, for example, after a time delay. In this embodiment, electrical stimulation is delivered to the chamber via a pair of stimulating electrodes, which elicits a response in the object. This response is measured by the passive electrodes. It will be understood that adding a pair of stimulating electrodes (in this embodiment, the pair of stimulating electrodes is a reference electrode and a stimulating electrode) can be used to test multiple excitable cells / objects and experimental scenarios (e.g., drug testing and screening).

[0190] The following non-limiting examples and experimental results are described:

[0191] Figure 10a

[0192] Visual assessment was used to identify responses and non-responses to the glucose challenge, with three batches of islets tested for each glucose challenge protocol used. Differences in glucose-induced electrical burst activity and inactivity cycles were also visually assessed across protocols (3 mM to 15 mM, 5.5 mM to 15.5 mM, and 7.3 mM to 17.3 mM). For all protocols used across the three batches (3 mM to 15 mM, 5.5 mM to 15.5 mM, and 7.3 mM to 17.3 mM), we identified electrical responses or non-responses of islets to increased glucose concentrations. In electrophysiological experiments with the 3 mM to 15 mM protocol, islets were initially inactive and subsequently responded to glucose addition (see [link to electrophysiological study]). Figure 10b (This also shows the baseline response to 5.5 mM glucose and the enhanced response to 15 mM glucose (see [link]). II. Islet responses In continuous islets, electrical activity was observed upon treatment with tolbutamide, but no response was observed from any subsequent glucose-increasing protocol. At different time points, artifacts were observed in the gaps between recordings reflecting “hidden” effects from glucose additions of varying recording lengths.

[0193] III. Background electrical activity

[0194] First, the electrical activity of the islets was analyzed, followed by an assessment of the increase in electrical activity after glucose addition. For all islet batches (batch 1, batch 2, and batch 3), the electrical response (μV) was quantified using RMS output (as described above) before or after glucose addition, by evaluating 2-minute samples (for 10-minute recordings), 3-minute samples (for 20-minute recordings), or 5-minute samples (for 30-minute recordings). Only data from the islets, confirming their presence on the electrodes, were included in the dataset.

[0195] IV. Glucose-induced electrical activity

[0196] Before quantifying the islet electrical responses, they were first characterized as either electrically active or inactive. The presence of electrical activity was considered as the islets being active. The presence of inactive MEA channels provided a comparison between recorded activity and inactivity. No intermediate category of islets that were near death but active was observed (indicated by persistent activity not linked to glucose (non-glucose responders)). In batch 1, all islets (n=16) tested using glucose protocols from 7.3 mM to 17.3 mM showed electrical activity (100%). In batch 2, all islets tested using glucose protocols from 7.3 mM to 17.3 mM (n=8) and from 3 mM to 15 mM (n=16) were electrically active (100%). From batch 3 (n=24), all islets tested using protocols from 5.5 mM to 15.5 mM showed electrical activity (100%). These results indicate that some electrical activity can be detected in the islets even at lower glucose levels or in control glucose.

[0197] Methods of manufacture and use

[0198] The ability of the RMS function to reflect changes in electrical activity associated with increased glucose concentration has been validated; it was used to determine whether electroactive islets respond to glucose and whether they are viable or unresponsive. Of the islets (n=16) tested in the 7.3 mM to 17.3 mM regimen in batch 1, 9 showed an increase in RMS. In the islets tested in the 7.3 mM to 17.3 mM regimen (n=8) and the 3 mM to 15 mM regimen (n=16) in batch 2, increases in RMS values ​​were observed after the glucose challenge. In the 5.5 mM to 15.5 mM regimen used in batch 3, 16 of the 24 islets tested showed increased RMS values. This presents the total number of islet regimens with increased RMS values ​​of 68.75%, 45.83%, and 66.66% for the 3mM to 15mM, 7.3mM to 17.3mM, and 5.5mM to 15.5mM regimens, respectively (see Figure 7d). One islet was tested using the 3mM to 15mM regimen from batch 2, showing a 3% marginal increase in RMS value, which was not classified as a glucose response. Those classified as unresponsive to glucose showed no change or a decrease in RMS value when glucose concentration increased. It was then determined whether the increase in RMS value was statistically significant when challenged with increased glucose concentrations. Only RMS values ​​of islets showing an increase were included. When present in culture medium, the mean RMS value (n=11) of islets tested using the 7.3 mM to 17.3 mM protocol showed a significant increase between 7.3 mM and 17.3 mM glucose concentrations (paired t-test, t=4.625, df=10, p=0.0009). At 7.3 mM glucose concentration, the mean (±SEM) RMS of the islets was 3.95 ± 0.72 μV, which significantly increased to 10.13 ± 1.82 μV as the glucose concentration increased to 17.3 mM. The RMS values ​​of the electrical response spanned 8.87 μV and 21.9 μV for 7.3 mM and 17.3 mM glucose, respectively. Therefore, most of the islets tested responded to the increase in glucose with a measurable electrical response and could be detected by measuring the RMS noise.

[0199] V Islet batch survival rate

[0200] The survival percentage for each batch was calculated by identifying islets that met the electroactive criteria and by observing the increase in RMS values ​​observed after glucose challenges using glucose protocols of 3 mM to 15 mM, 5.5 mM to 15.5 mM, and 7.3 mM to 17.3 mM. Islets were characterized as having survival rate because the increase in RMS values ​​in response to 7.3 mM to 17.3 mM, 3 mM to 15 mM, and 5.5 mM to 15.5 mM was significant. This resulted in the following batch survival rates: 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). Survival percentages varied depending on the date of testing. In batch 1, 15 islets were tested after 3 days of incubation, with 9 islets (60%) showing a survival rate (indicating an increase in RMS). After 4 days of incubation, 1 islet tested showed no survival (indicating a decrease in RMS). In batch 2, 15 islets were tested after 1 day of incubation, with 4 islets (26.66%) showing a survival rate. After 2 days of incubation, all 6 islets tested showed a survival rate (100%). After 6 days of incubation, another 3 islets were tested, with all 3 showing a survival rate (100%). Starting with batch 3, 5 islets were tested after 1 day of incubation, with 3 showing a survival rate (60%). After another two days of incubation, this increased to an 80% survival rate (4 out of 5 islets). After 5 days of incubation, this decreased to 61.5% (8 out of 13 islets). Therefore, using a combined chip to rapidly measure electroactivity distinguishes between islet batches and islet survival rate.

[0201] This disclosure demonstrates that the islet electrical activity of each islet isolate varies from batch to batch, and that impairing islet function using prolonged hypoxia results in a lack of responsiveness to glucose challenges, thus reflecting islets with no viability. These two observations indicate that electrical activity in response to glucose challenges is a good representative of the health and function of islet batches for rapid determination. This novel device successfully captures islets in a manner that facilitates multichannel recording and simultaneous interrogation of multiple islets. It is envisioned that this will form the core technology for new benchtop devices to determine islet quality prior to transplantation. Such a device can be used in every islet transplantation laboratory worldwide. This will also provide standardization for islet assessment and enable comparisons from different laboratories. Furthermore, the device, along with improved isolation and processing techniques, ultimately leads to an effective technique for functional classification from non-functional islets. With the development of human embryonic stem cell-derived islets in early phase 1 clinical trials in humans, there is also an objective need to assess the viability of such stem cells, and the potential use of such instruments in this field is also possible. In addition, this device can be used for cell clusters from other tissue sources to test viability.

[0202] According to an embodiment, the following comments are provided regarding the details of the materials and manufacturing methods.

[0203] Figure 6b

[0204] I. Design of Microelectrode Arrays (MEAs)

[0205] The custom MEA was designed using AutoCAD (Autodesk, Inc., California, USA). Several different electrode configurations were found to be suitable (see Figure 6A for example). The design in Figure 6A is characterized by a total of 11 recording electrodes and a uniform ground reference electrode (details of which can be found in...). II. Manufacture of microelectrode arrays (MEAs) The recording electrode has a diameter of 30 μm, and the distance between the recording electrode and the ground electrode is 50 μm. The AutoCAD design was sent to Micro Lithography Services Limited (Chelmsford, UK) for fabrication into a thin film or glass photomask.

[0206] Spin coating of photoresist

[0207] The MEA was fabricated in the Nanofabrication Laboratory at Heriot-Watt University. Borosilicate was used as the substrate (h in Figure 6). The borosilicate was cut to a measurement of 49x49 mm using a DAD3220 wafer dicing saw (DISCO Corporation, Tokyo, Japan). Photolithography was used to transfer the electrode design and insulation pattern onto the borosilicate. The electrodes and electrode tracks are titanium (g in Figure 6) deposits, while the track insulation, excluding the electrode tips and pads, is a SiO2 deposit (f in Figure 6).

[0208] III. Alignment and UV exposure

[0209] Prior to deposition, the borosilicate substrate was washed with acetone and diluted with Decon 90 (Decon Laboratories Ltd, East Sussex, UK). It was then rinsed with deionized (DI) water and dried using a filtered air gun. This was to ensure the borosilicate surface was dust-free and completely clean before starting the process. The borosilicate preform was then placed on a spin coater SPIN 150 (SPSEurope, Putten, Netherlands) and held in place by vacuum sealing. A negative photoresist AZnLOF 2070 (MicroChemicals, Ulm, Germany) was carefully applied to the entire surface of the borosilicate to avoid air bubbles. It was then spin-coated at 3500 rpm for 40 seconds. Once spin-coated, the negative photoresist, which typically forms a 7 μm thick layer, was pre-diluted to form a 1 μm layer. Following this process, the substrate is hard-baked at 95°C for 3 minutes.

[0210] IV. Developing

[0211] The next step involves aligning one of the multi-electrode design thin-film photomasks with the fabricated substrate. This is done using an MJB3 mask aligner (MJB3 Mask Aligner, S.SS MicroTec, Garching, Germany). The thin-film photomask is placed on a mask holder, and then loaded and secured to the aligner. The substrate is positioned on the substrate holder, which is held in place using vacuum suction, and then slid under the photomask. The substrate is brought into contact with the thin-film photomask, and then a release lever is manipulated to align the design with the substrate. Alignment is achieved by adjusting the x and y axes in micrometers. Once aligned, the release lever is pushed back, returning the substrate to contact with the thin-film photomask. The substrate is then UV exposed for 40 seconds, and once exposure is complete, the substrate is carefully removed from the mask aligner. A post-exposure bake at 115°C for 90 seconds is then performed.

[0212] V. Electrode deposition

[0213] The substrate was then developed using AZ 726 MIF (metal ion-free) developer (Microchemicals). It was initially immersed in the developer for 60 seconds, and then closely monitored for 15 to 30 seconds until the design features became visible. The substrate was then washed in diluted Decon 90 for approximately 30 seconds, followed by rinsing with deionized double-distilled water and drying with a filtered air gun.

[0214] VI. VII. Second spin coating

[0215] Once the substrate is deposited, titanium is deposited using a technique called physical vapor deposition (PVD) on a device called the "Minilab 080" (Moorfield Nanotechnology, Knutsford, UK). This involves directing an electron beam onto a metal, which then evaporates and deposits onto the substrate. A borosilicate substrate is placed in an outer chamber with the side to be coated facing downwards. The outer chamber must be degassed to achieve the same vacuum level as the main chamber, which contains the titanium-filled crucible, before the substrate is slid into the main chamber. During this process, the required parameters are input into an SQM-160 rate / thickness film deposition monitor (Inficon, Bad Ragaz, Switzerland). These include the thickness of the metal layer to be deposited, the density of the metal to be deposited, and the Z-ratio. The thickness is set to 10kJ (1000nm), the titanium density to be 4.500, and the titanium Z-ratio to be 0.628. Once the substrate is secured and the main chamber door is locked, the electron beam is directed onto the titanium using a controller knob. The power is slowly increased to up to 100 mA before opening the shutter to allow titanium to evaporate onto the borosilicate substrate. The rate is monitored and maintained between 2 and 3 s / s. The shutter closes automatically, and the electron beam shuts off when the titanium layer reaches 1000 nm. The substrate is then carefully removed from the main chamber into an outer chamber, which is then pressurized to room temperature before being opened to collect the substrate. Electrode deposits have formed a titanium layer across the entire surface of the borosilicate. Therefore, the next step involves removing excess titanium to leave only the array design. This is done by dissolving the previously applied negative photoresist using a TechniStrip NI555 (MicroChemicals) at 80 °C until all unwanted titanium layers are removed. Dissolving the negative photoresist means removing the titanium layer deposited on top of it, leaving only the titanium array design.

[0216] VIII. Insulator deposition

[0217] The next step is to prepare the surface for depositing the silicon dioxide (SiO2) insulating layer. The photolithography process is similar to that described in the previous sections. However, this time an insulating photomask is used, and AZ 1505 positive photoresist (MicroChemicals) is used. The spin coater is set to 2000 rpm for 30 seconds. Furthermore, the substrate is developed in AZ351B developer MIC (containing metal ions - MicroChemicals), which is diluted 1:4 (1 part developer and 4 parts DI water). Afterward, the substrate is washed as described previously, and SiO2 deposition is performed.

[0218] IX. Alternative laser lithography

[0219] The same process as for titanium was used to deposit SiO2; however, the density and Z-ratio parameters were changed to 2.648 and 1.000, respectively. The SiO2 thickness was 500 nm. After deposition, the next step was to remove the SiO2 from the electrodes and contact pads, as these were the areas that needed to be exposed. In this case, acetone was used to remove the positive photoresist, leaving SiO2 only on the tracks (the tracks that connect the electrodes to the contact pads).

[0220] Design and manufacture of microfluidic microchannels

[0221] After testing these custom MEAs, it was discovered that the tracks were not always fully insulated, or the electrodes were not exposed. These problems were addressed by using a DWL 66+ (Heidelberg Instruments Electromechanical GmbH, Heidelberg, Germany) 2.5 D laser lithography system. This machine is capable of laser etching structures down to 300 nm and can perform direct photoresist patterning. Therefore, this means that the steps of aligning the thin-film photomask and using UV exposure are no longer required. The array design was adjusted in KLayout (Matthias K. Fellerin, Germany) for reading via laser writer software. A borosilicate substrate previously spin-coated with a negative photoresist layer was placed on the platform, and the remainder was completed using a software package provided by Heidelberg Instruments. The write head automatically aligns itself with the substrate before it begins etching the array design onto the photoresist. Once this is complete, the same steps described previously for titanium deposits are performed; however, this time, the titanium thickness is set to 150 nm. The insulation design is also laser-etched instead of using a thin-film photomask. This means that the electrodes and contact pads are precisely removed, and thus, the electrodes and contact pads are exposed after deposition (100nm) and after the SiO2 is removed.

[0222] I. Microchannel manufacture using soft lithography

[0223] Figure 1

[0224] The microchannel was designed using Adobe Illustrator (Autodesk), and then sent to Micro Lithography Services to be manufactured into a thin-film photomask. The design is characterized by… Figure 2 , Figure 3 , Figure 4 , II. Spin coating and UV exposureAs shown in Figures 1 and 5. The soft lithography scheme for fabricating the PDMS device (MicroChem, 2015) is similar to the lithography scheme used when fabricating a custom MEA, but with different parameters. A test-grade silicon wafer (SILI-0005, PI-KEM Ltd.) with a diameter of 7.62 cm and a thickness of 380 μm + / - 50 μm was used instead of a borosilicate substrate.

[0225] III. Developing

[0226] A silicon wafer was placed on a spin coater (WS-650MZ-32NPP, Laurell Technologies Corporation) and held in place by vacuum. A negative photoresist, SU-8 2025 (Microchem), was spin-coated onto the wafer in two stages. In the first stage, the spin speed was 500 rpm for 10 seconds with an acceleration of 100 rpm / s, and in the second stage, the spin speed was 3000 rpm for 39 seconds with an acceleration of 300 rpm / s. These settings yielded a film thickness of 100 μm. This process was repeated three times to obtain thicknesses of approximately 250 μm to 300 μm. The wafer was then soft-baked at 65°C for 1 minute, followed by a soft-baked period at 95°C for 5 minutes, and then returned to 65°C for another minute. A thin-film photomask with the design was then carefully placed on top of the wafer, ensuring it did not move once in contact with the wafer. The wafer was then exposed to UV light for 41 seconds, followed by exposure-to-bake at the same temperature and time as soft bake. The transparent portions of the photomask (the two compartments and the microchannels) became cross-linked, while the dark portions of the photomask did not cross-link.

[0227] IV. PDMS preparation

[0228] The wafers were immersed in SU-8 developer and sonicated for 60 seconds. They were then washed with isopropanol and dried with an air gun. The wafers were placed back in the developer for another 60 seconds, but without sonication. The same washing steps were performed until the wafers were clean. If any streaks were still visible, the wafers were placed back in the developer for another 60 seconds, and this process was repeated until they were no longer present. Ultimately, as long as the wafers could remain intact, they could be used as molds to create microchannels. The surface of the wafers is hydrophilic, meaning it is difficult to peel PDMS from the wafer surface without leaving any residue or damaging the wafer. One drop of trichloro(1H,1H,2H,2H-perfluorooctyl)silane (448931, Sigma-Aldrich, Missouri, USA) was added to the wafers and evaporated at room temperature in a fume hood, ensuring the top of the culture dish was sealed. The purpose was to make the surface hydrophobic and thus avoid the problems mentioned above. The wafers were held in a glass culture dish, and aramid zeolite was used to bond the wafers to the culture dish.

[0229] PDMS / electrode array device assembly

[0230] PDMS was prepared by mixing Sylgard 184 siloxane elastomer matrix with a curing agent at a ratio of 10:1. The PDMS was then poured over a wafer, and air bubbles were removed by placing it in a vacuum chamber before curing. The PDMS cured at 60°C for 1 hour. Once cured, the PDMS modules (microchannels) were carefully removed using a scalpel, attempting to avoid damaging any features on the wafer. Access holes were then cut on either side using 1mm and 6mm biopsy punches to form vias. This was also done carefully to avoid damaging the microchannels.

[0231] I. Islets

[0232] The fabricated PDMS modules were then integrated into a custom MEA. This was achieved by placing the PDMS modules and bonding them to the electrode surfaces in a Zepto plasma system (Diener Electronic, Ebashausen, Germany). The surfaces were exposed to 20% O2 plasma for 5 minutes under vacuum. Before integrating the PDMS microchannels into the MEA, the PDMS was carefully aligned with the ground and recording electrodes. This was done manually using an inverted microscope (see Figure 6D).

[0233] The following non-limiting comments are provided regarding the use and verification of the device:

[0234] II. Electrophysiology solutions

[0235] Islets of Langerhans are isolated from donor pancreas using enzymatic and mechanical methods, with the final step being the purification of the isolated islets from exocrine tissue (Matsumoto et al., 2007).

[0236] III. Electrophysiology setup

[0237] Medium containing 7.3 mM glucose was used as the starting condition for electrophysiological experiments. An additional 10 mM glucose (totaling 17.3 mM) was used to supplement the medium, which was then used as a hyperglycemic solution. The solution was also optimized to provide a "cleaner" electrophysiological recording by using an islet solution comprising 138 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, CaCl2, 5 mM HEPES, and pH 7.4 (Kindmark et al., 1994). For hypoglycemic conditions (control) and hyperglycemic conditions, the islet solution was supplemented with 3 mM and 15 mM glucose, respectively.

[0238] IV. Electrophysiology protocol

[0239] Field potential recordings were acquired using an Axon CNS Digidata 1440A digitizer (Molecular Devices, California, USA), a 16-channel microelectrode amplifier, a 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 pancreatic islet electrical activity. This protocol was extended to 30 minutes. A 10x head stage (Omnetics, Minnesota, USA) was used to pre-amplify the electrical activity signal. Recording settings for the high-pass and low-pass filters were 3Hz and 5kHz, respectively. The gain was set to 2000x, GND was selected, and a notch filter was enabled to suppress line-dependent noise at 50Hz / 60Hz.

[0240] V. Signal amplification

[0241] Islets were used for experiments incubated in culture medium for 1 to 6 days. 200 μl of sample from a 5 mL aliquot was transferred to the inlet channel of the microfluidic channel, and the islet positioning relative to the electrodes within the microfluidic channel was examined. 7.3 mM culture medium was used for initial loading. This prevented the islets from adhering to the walls of the microfluidic channel. For experiments with 3 mM to 15 mM glucose, after addition to the MEA device, the remaining culture medium was removed and replaced with 200 μl of 3 mM glucose islet solution. Once positioning was determined (via an inverted optical microscope), the islets were incubated in solution at room temperature for 10 minutes before recording. The MEA / microfluidic device was connected and placed under an inverted microscope, and recordings were performed for 5 minutes and 10 minutes, respectively, before adding 17.3 mM glucose culture medium and 15 mM glucose islet solution. Addition was performed by removing the remaining initial solution from the tip of the inlet pipette (via a micropipette) and adding the additional glucose solution via a syringe. Reconfirm the location using an inverted microscope and continue recording for 15 or 20 minutes. Islets from batch 3 were evaluated using a 5.5 mM to 15.5 mM culture medium protocol, with five-minute runs recorded before and after glucose addition. A total of 7 records were taken from batch 1, 11 from batch 2, and 19 from batch 3. Of these records, 11 were performed using the 7.3 mM to 17.3 mM glucose protocol, 8 using the 3 mM to 15 mM protocol, 14 using the 5.5 mM to 15.5 mM protocol, and 5 treated with tolbutamide (50 μM). Glucose concentrations of 3 mM, 5.5 mM, and 7.3 mM, and tolbutamide were used as controls. 15 mM, 15.5 mM, and 17.3 mM glucose were used as elevated concentrations. After each experiment, the MEA / microfluidic device was rinsed with deionized water before starting a repeat experiment. All experiments were conducted at room temperature (20°C).

[0242] VI. Signal digitisation

[0243] Model 3600 amplifiers were purchased from AM Systems (Washington, USA) at a fraction of the cost of commercial array amplifiers. The Model 3600 amplifier features 16 channels, 11 gain settings ranging from 2x to 20,000x, 8 low-pass filters ranging from 100Hz to 20kHz, 8 high-pass filters ranging from 0.3Hz to 500Hz, and a notch filter (50Hz or 60Hz) for each channel. The signal was pre-amplified by a 10x head stage, which was then pre-amplified by a x10 head stage (Omnetics, Minnesota, USA) located next to the array, before the data was passed to the Model 3600 amplifier. A device was fabricated in the electronics workshop that flexibly holds the MEA and connects it to the preamplifier head stage. It consists of a retaining plate and a top plate, the top plate including spring pins (PD8JS-2.2, Coda-Systems, Essex, UK) with a 1mm tip diameter and 0.45mm working stroke for mating with the MEA's electrode contact pads. With the assistance of the electronics workshop, a total of three recording units (RD1, RD2, and RD3) were manufactured, each with a different arrangement of spring pins and unit-to-amplifier connectors to enable recording for all 59 electrodes of a commercial MEA and to allow recording and stimulation when using a custom MEA. Pins are connected to the preamplifier headstage via nanoribbon connectors (NPD-18-WD-18.0-C-GS, Omnetics). Each cable is soldered to a single pin, except for the ground and 10x reference cables, both of which are soldered to a single ground pin.

[0244] VII. RMS quantification method

[0245] The analog signal from the amplifier was digitized using a digital data acquisition system (PCe-6343, National Instruments, Texas, USA). The digital signal was then viewed in real time using Clampex (Molecular Devices, California, USA). All 16 channels could be viewed simultaneously in Clampex.

[0246] https: / / citregistry.org / system / files / 10AR_Scientific_Summary.pdf

[0247] The sensitivity of the root mean square variance (RMS) of the signal to changes in the electrical response was verified by separately calculating the RMS of three separate records from the islets of Langerhans in glucose protocols of 7.3 mM to 17.3 mM, 3 mM to 15 mM, and 5.5 mM to 15.5 mM from batches 1, 2, and 3, respectively. The RMS was calculated in 1-minute increments in the records, and the records were plotted to determine whether changes in RMS were synchronous with changes in electrical activity. RMS measurements were obtained using Clampfit's power spectrum function (version 10.7), where the window was set to no signal change (rectangular), output as an average spectral segment, maximum length, spectral resolution (spectral bin width) of 0.038147 Hz, and the RMS measurements and plots were set to exclude the first spectral bin (default setting). All RMS measurements in the electrical response are in microvolts (μV) (see Figure 7).

[0248] According to an embodiment, a combination of microfluidic channel design and trap or confinement is integrated with a printed microelectrode array, as well as input and output chambers. The channel includes a microfluidic bypass that allows solution flow once the islets have been attached to the trap. This flow system allows for rapid exchange of a normal storage solution (5.5 mM glucose) with a storage solution containing a higher concentration (15.5 mM glucose). The change in glucose concentration causes a measurable electrical change in the islets, detected by a reader. This activity pattern is only observed in fully functional islets. Multiple channels (1, 8, and 16 in our current device, but potentially up to 64 in future developments) enable rapid determination of the percentage of functional islets in a sample. This device allows for rapid, accurate, and label-free assessment of islet health. It will be used to objectively assess islet viability and enable the use of fewer but dissected islets during surgery, even in cases where ischemic time is prolonged. This will enable the use of more materials and more patients for life-saving transplants, resulting in reduced waiting list numbers and waiting times. Furthermore, due to the urgent need for improved islet maintenance, sorting, and quality control methods, this (multi-device) system will also provide greater efficiency at all levels.

[0249] According to an embodiment, islet transplantation is performed in recipients with type 1 diabetes using islets isolated from a donor pancreas, and the field of stem cell-derived human islets is a focus of in-depth research. It is a life-saving treatment that stabilizes blood glucose control and can even lead to insulin independence. However, transplantation outcomes can be highly correlated with the number of islets and their survival rate. The transplantation process is a multi-step procedure involving pancreas harvesting, tissue dissociation, islet purification, cell culture, and islet transplantation via the portal vein into the recipient's liver. Islet loss occurs at each step (primarily due to ischemia). Less than 50% of the islets from a single pancreas are isolated (~500,000 islets), and of these islets, it is estimated that less than 50% are transplanted into the liver, where the recipient requires islets from 2 to 3 donor pancreas. Prior to transplantation, there is no rapid method to establish islet quality in a sample. This device provides core technology to enable rapid estimation (%) of functional islets in a sample. It will meet the need by measuring the electrical activity of the islets as a representative of pancreatic health. The design of the microfluidic delivery system and the electrical interface (electrode array) together provides a "hands-free" module for measuring islet electrical function. Uniquely, when an islet sample is pipetted into a 200µL holder, the islets enter the system and are captured above the electrodes by capillary and gravity-driven flow. The unique channel design means that an additional 200µL solution containing elevated glucose can be pipetted into the delivery system to generate an electrical response in live islets. This unique combination (connecting the bypass system to the electrodes in the microelectrode module) means that after a measurement has been performed, the sample can be rinsed and repeated with additional samples.

[0250] The above description of the specific embodiments is by way of example only. Those skilled in the art will understand that changes can be made to the described embodiments without departing from the scope of the invention.

[0251] References

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Claims

1. A microfluidic device, comprising: Microfluidic module, the microfluidic module comprising: - Multiple fluid paths, said multiple fluid paths being located between the input and output sections; and - Multiple limiting elements, wherein each limiting element is disposed on a corresponding fluid path in the multiple fluid paths, wherein each limiting element 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 their respective fluid path at a corresponding sensing area; - At least one additional fluid path between the input and the additional output, wherein the at least one additional fluid path is in fluid communication with the plurality of restraints, and wherein the additional fluid path includes a delivery portion and an output portion, the delivery portion being between the input and the plurality of restraints to allow additional fluid to be delivered to the captured and / or restrained one or more objects, and the output portion allowing the additional fluid to be removed from the additional output; The device further includes: - An electronic sensing module, the electronic sensing module comprising: --At least one sensing element, the at least one sensing element being arranged to sense one or more signals from the respective sensing areas of the plurality of constraints.

2. The apparatus according to claim 1, wherein, The object includes at least one of the following: organoid, spheroid, tissue spheroid, pancreatic islets, cell or tissue object.

3. The apparatus according to any one of the preceding claims, wherein, The object includes tissue spheroids, optionally, islets of Langerhans derived from the pancreas.

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

5. The apparatus according to any one of the preceding claims, wherein, The output portion of the additional fluid path is downstream of the delivery portion and the plurality of limiting elements.

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

7. The apparatus according to any one of the preceding claims, the apparatus comprising a plurality of channels, the plurality of channels comprising: A first channel, the first channel being between the input section and the additional output section, the first channel defining the additional fluid path between the input section and the additional output section; A second channel is connected to the plurality of limiting members and the output section, wherein the second channel is connected to the first channel via the plurality of limiting members, 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 section and the output section.

8. The apparatus according to any one of the preceding claims, wherein, The first and second channels are sized to allow fluid flow through the object, and the limiting element includes an opening between the first and second channels, the opening being sized to prevent the object from passing through.

9. The apparatus according to any one of the preceding claims, wherein, The plurality of limiting elements includes 5 or more, optionally at least 10, optionally at least 15, optionally at least 60, optionally at least 100 limiting elements.

10. The apparatus according to any one of the preceding claims, wherein, The plurality of restrictors are arranged at the overlap of the plurality of fluid paths and the additional fluid path to allow objects to be sequentially conveyed to the restrictors, such that when a restrictor is blocked by an object, another object in the fluid advances to a subsequent restrictor and / or toward the additional output.

11. The apparatus according to any one of the preceding claims, wherein, The at least one sensing element is disposed on a layer below the plurality of restrictors, optionally such that an object that is at least partially restricted and / or captured comes into contact with the at least one sensing element.

12. The apparatus according to any one of the preceding claims, wherein, The at least one sensing element includes a plurality of sensing elements, the plurality of sensing elements including at least one reference electrode and one or more sensing elements for each constraint, and / or wherein the at least one sensing element is configured to sense electrophysiological signals and / or signals in response to the electrophysiological activity of the object.

13. The apparatus according to any one of the preceding claims, wherein, The sensing module includes: One or more stimulating elements, such as stimulating electrodes, configured to generate electronic stimulation, such as a stimulation signal; and One or more sensing elements, such as sensing electrodes, are configured to sense a response to the electronic stimulation, optionally wherein the one or more stimulating elements and / or sensing elements are positioned to stimulate and / or sense activity in the sensing region.

14. The apparatus according to any one of the preceding claims, wherein, The at least one sensing element is configured in the electrode array to be aligned with and / or set as a part adjacent to the at least one limiting element.

15. The microfluidic device according to any one of the preceding claims, wherein, The device further includes a reference sensing element, optionally wherein the reference sensing element is disposed at or across at least a portion of the plurality of fluid paths, and / or at or across at least a portion of another fluid path.

16. The apparatus according to any one of the preceding claims, wherein, Each of the limiting members includes an opening whose cross-sectional area is at least 50%, 60%, 70%, 80%, 90%, or 95% smaller than the diameter of the one or more objects of interest.

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

18. The microfluidic device according to any one of the preceding claims, wherein, The plurality of fluid paths and the additional fluid paths are formed by a plurality of channels, wherein at least one of the width and / or height of at least one channel is selected from a range, thereby increasing or decreasing the fluid flow rate.

19. An apparatus comprising the means according to any one of claims 1 to 18, and further comprising processing resources configured to: Receive sensor signals and / or data representing the sensor signals from the at least one sensing element; Process the data and / or signals to determine at least one of electrical activity, survival rate, function, and / or response to stimuli, optionally Electrical stimulation is provided via one or more stimulating electrodes.

20. The device according to claim 19, wherein, The processing resources are configured to determine at least one of the following: a) Measurement of electrical activity, said electrical activity in response to electrical stimulation and / or additional fluid, optionally wherein, The additional fluid contains chemicals and / or drugs, such as a glucose solution; b) The number of viable objects, and / or functional tissue objects, and / or multicellular objects in the sample; c) Perform diagnostic and / or survival indications based on electrical activity sensed at the plurality of restraints and / or sensing areas, for example, wherein the diagnostic and / or survival indications may be based on electrical activity in response to exposing the object to an additional substance or electrical stimulation, the additional substance optionally comprising chemicals and / or drugs, such as glucose.

21. A method for analyzing a plurality of objects using the apparatus according to any one of claims 1 to 19, said plurality of objects being, for example, a plurality of tissue objects and / or multicellular objects, the method comprising: Multiple objects in a fluid are provided to the input of the microfluidic device, wherein the fluid initially flows through the device along the multiple fluid paths until the multiple objects reach the multiple restraints and are captured and / or at least restrained by the restraints; The at least one sensing element is used to sense at least one sensor signal from the plurality of objects.

22. The method of claim 21, further comprising: Additional fluid is delivered via the input section to the plurality of objects that are captured or at least confined within the plurality of restraints via the at least one additional fluid path.

23. The method according to claim 21 or 22, further comprising: Electrical stimulation is delivered to the plurality of objects that are captured or at least confined within the plurality of restraints.

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

25. The method according to claims 21 to 24, wherein, The additional fluid includes at least one of the following: a) A washing solution used to remove any solution via the additional output section; b) Glucose solution; c) One or more drugs, and / or cell, and / or object activity inhibitors; d) One or more molecules used to label the object; e) A higher concentration than that of the fluid containing the plurality of objects.

26. The method according to any one of claims 21 to 25, wherein, The method further includes isolating the output electrical signal from the captured object for the plurality of limiting elements of the microfluidic device.

27. The method according to any one of claims 21 to 26, further comprising: Process the sensor signals and / or data representing the sensor signals to assess at least one of cellular electrical activity, extracellular potentials, field potentials, survival rates, functions, and / or responses to stimuli.

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