STIMULATED MUSCLE CELLS

Biological systems need to extract energy from their environment to stay alive. When cells lose the ability to conserve energy from dietary nutrients, both their function and survival are compromised. Bioenergetic failure is often linked to altered mitochondrial function and underpins medical disorders including neurodegeneration, inflammation, infectious disease and cancer, as well as the Metabolic Syndrome, a group of disorders that collectively increase the risk of developing type 2 diabetes and cardiovascular disease. It is becoming increasingly clear that decreased 'bioenergetic health' is an early warning sign of cellular dysfunction and consequent disease [ I ]. Cellular bioenergetics can be measured readily in real time and in intact cells by extracellular flux (XF) analysis. This technology was developed by Seahorse Bioscience (now part of Agilent Technologies) and allows functional measurement of energy metabolism by non-invasive fluorescent detection of metabolites (oxygen and protons) that are taken up or released by cultured cells [US2007008740 I ].

The ability to monitor bioenergetic function through XF analysis has clear potential for diagnosing disease, identifying new drug targets, and for detecting off-target effects of compounds with therapeutic potential.

XF technology has been used to study involvement of bioenergetic failure in a wide range of diseases and has been described extensively in both practical [2, 3] and theoretical [4] contexts. Translational potential of XF assays, and indeed of in vitro assays generally, depends on how closely one is able to approximate the physiological and pathological conditions that cultured cell models face in vivo. In the case of studies on skeletal and cardiac muscle, it is important to study muscle cells that are able to contract.

In bioenergetic studies on resting skeletal muscle [5,6], rat (L6) and human myoblasts were differentiated to myotubes by growing them at a low serum concentration for 8 days. L6 myotubes start to contract spontaneously when grown for longer ( 1 0- 1 3 days), and align into myotube bundles when left for 2-3 weeks. These bundles contract both spontaneously and when stimulated electrically [7].

I Extended growth on XF plates has enabled measurement of mitochondrial activity of spontaneously contracting L6 myotubes in real time and comparison with the activity of resting myoblasts (Fig. I ). Technology has been developed by the lonOptix corporation that allows electrical stimulation of contractile cells during cell culture [US7 I 48059], but the smallest culture platform that is able to accommodate this C- PACE technology is a 12-well plate. XF assays are run on plates with well surfaces that are much smaller, and analysis of skeletal muscle bioenergetics is thus limited to resting and spontaneously contracting myocytes at present. The XF data shown in Figure I reflect oxygen uptake by resting L6 myoblasts and spontaneously contracting L6 mytobes. These data are the means ± SEM of 5 and 7 separate experiments with myotubes (blue symbols) and myoblasts (red symbols), respectively. Cellular respiration in the cumulative presence of 5 g/mL oligomycin, I μΜ uncoupler: N ⁵ ,N ⁶ - f)/ ^' s(2-fluorophenyl)-[ 1 ,2,5]oxadiazolo[3,4-b]pyrazine-5,6-diamine BAM 15 (uncoupled), and a mix of I μΜ antimycin A and I μΜ rotenone (non-mito) was normalised to basal respiration. Myotubes and myoblasts respond to these respiratory effectors in a similar way, in so much that inhibiting ATP synthesis with oligomycin and uncoupling oxidative phosphorylation with BAM 15 decreases and increases oxygen uptake, respectively. The mitochondrial electron transfer inhibitors antimycin A and rotenone largely abolish respiration in both systems, and remaining oxygen consumption arises from non- mitochondrial processes. This typical respiratory behaviour allows calculation of coupling efficiency of oxidative phosphorylation (part of mitochondrial respiration used to make ATP), a bioenergetic parameter that is highly sensitive to changes in energy metabolism. Coupling efficiency (Fig. I , inset) was calculated as the % of basal mitochondrial respiration that was oligomycin-sensitive, and appeared significantly lower in myotubes than myoblasts (P < 0.0001 ). Low coupling efficiency is likely owing to the comparably high work output demanded from contracting myotubes, similar to the low fuel efficiency of a car engine running at high speed. Described herein is a cell-based assay that allows electrical control over contractile workload of skeletal and cardiac muscle cells during real-time measurement of their bioenergetic behaviour. Figure 2 demonstrates that electrical stimulation increases the rate of both oxygen uptake and proton production by L6 myotubes. Such XF changes are consistent with increased energy demand from muscle contraction: because ATP flux in healthy muscle is largely demand-driven, a need for energy will stimulate ATP synthesis. Increased oxygen uptake and medium acidification suggest strongly that both mitochondrial and glycolytic ATP synthesis rates are increased by electrical stimulation.

An aspect of the present invention cell culture plate stimulator system for measuring bioenergetic behaviour of muscle cells, the system comprising a plate having one or more wells in which muscle cells can be grown, and means for applying, in use, a pulsed electrical field to the cells to stimulate their contraction in use, in which the means for applying the pulsed electrical field comprise one or more H-bridge circuits.

The field may be applied to the or each well by one or more electrodes.

The or each electrode may be carbon or carbon-based.

The height of the conductive surface of the electrode/s may be I mm or less.

The or each electrode may be formed integrally in the base of the or each well. In some embodiments a plurality of wells are provided.

The means for applying a pulsed electrical field may apply electrical pulses of substantially equal proportion to all wells. The plurality of wells may be arranged in a plurality of ranks and in which a dedicated H-bridge controls electrical field distribution in each rank.

A dedicated H-bridge may be assigned to each well. The means for applying a pulsed electrical field may generate an electrical field distribution to stimulate cells uniformly across the well/s. A further aspect provides an extracellular flux microplate for the real-time measurement of cellular bioenergetics, the microplate comprising carbon or carbon- based electrodes for applying, in use, a pulsed electrical field to each of a plurality of wells.

A further aspect provides a cell culture stimulator for extracellular flux plates, the stimulator comprising a control circuit having one or more H-bridges for applying an electrical field to plate wells in use. A further aspect provides an in vitro system for electrically stimulating skeletal muscle cells, comprising a microplate with a plurality of wells in which contractile skeletal muscle can be grown, and means for applying a pulsed electrical field to the cells to stimulate their contraction, and means for measurement of extracellular flux in real time, whereby the bioenergetic behaviour of the cells can be analysed and quantified in real time whilst they are contracting.

The system may be configured for pacing muscle cells in an extracellular flux analyser. The means for applying a pulsed electrical field may comprise a printed circuit board.

A further aspect provides an in vitro cell-based assay allowing electrical control over the contractile workload of muscle cells during real-time measurement of bioenergetic behaviour, the assay comprising:

providing an extracellular flux microplate, the microplate comprising means for applying, in use, a pulsed electrical field to each of a plurality of wells;

growing muscle cells in the wells;

providing an extracellular flux analyser;

providing a stimulator comprising a printed circuit board having an H-bridge control circuit for applying an electrical field to the plate wells in use;

- stimulating the muscle cells in the wells; and

conducting extracellular flux measurement. The extracellular flux measurement may comprise oxygen consumption and/or acid production.

Some embodiments relate to the bioenergetic behaviour of cardiomyocytes and characterised by quantification of glycolytic and mitochondrial ATP synthesis fluxes from oxygen uptake and medium acidification data that may be obtained from conventional XF analysis.

Some aspects of the present invention relate to a cell-based cardiotoxicity assay.

In some embodiments existing extracellular flux (XF) technology is adapted to allow electrical stimulation of mammalian cells during real-time measurement of their bioenergetic function. Such electrical pacing offers user-control over the contractile activity of skeletal and cardiac muscle cells thus closely mimicking the physiological workloads that these cultured cells face in vivo.

The present invention provides an apparatus and method that allow electrically stimulation of cells during XF analysis, and specifically relates to the electronic specifics that underpin the technology. Electrical stimulation of cells during XF analysis has been attempted before [8]. However, application of the reported method (using platinum electrodes and high-voltage ( 150V) stimulation) reveals technical issues relating to distorted electrical waveforms and a noisy signal that cause significant electrolysis, during and between electrical pulses, and consequent cell damage within minutes. In some aspects and embodiments the present invention relates to streamlining of electric stimulation of 24-well XF plates solving the technical issues mentioned above. Other aspects and embodiments relate to validation of the biological information gained from the XF-pacing assay. Electronic developments relating to XF-pacing assays formed in accordance with the present invention include the following, some or all of which may be included in various embodiments. Optimised circuit design to ensure that electrical pulses of substantially equal proportion are delivered to all of the 24 wells on the plate. A printed circuit board may be provided which can be mounted on an existing removable tray in the XF instrument.

Optimised electrode shape and carbon material, for example carbon fibre set in resin, glassy polymeric carbon and graphene. Metals such as platinum and gold may be included. Signal-generating and -amplifying circuitry to provide electrical contacts that will accommodate and connect electrically to XF culture plates with carbon electrodes built into their base. This adaptation will allow electrical stimulation of all wells via the bottom of XF plates. Optimised electric field distribution across the bottom of the wells to ensure all (or most) cells are stimulated uniformly. Some embodiments may use optimised circuitry during cell growth to improve cell alignment to the electric field. Contractile behaviour may be visualised microscopically so that contraction may be calibrated against applied voltage and frequency.

Different aspects and embodiments of the invention may be used separately or together.

Further particular and preferred aspects of the present invention are set out in the accompanying independent and dependent claims. Features of the dependent claims may be combined with the features of the independent claims as appropriate, and in combination other than those explicitly set out in the claims.

The present invention will now be more particularly described, by way of example, with reference to the accompanying drawings, in which:

Figure I - Oxygen uptake by resting L6 myoblasts (red) and spontaneously contracting L6 myotubes (blue) measured by XF analysis. Figure 2 - XF-pacing prototype data. Oxygen uptake (blue) and proton production (red) traces obtained from measuring the effect of electrical stimulation in a single XF24 well. A I OV-stimulation was applied at I Hz from time point A and the waveform frequency was increased to 5 Hz at point B. Stimulation was switched off at point C.

Figure 3 - H-bridge-based signal generator.

Figure 4 - Example of a circuit board.

Figure 5 - Example of system components forming part of an embodiment of the present invention.

Example embodiments are described below in sufficient detail to enable those of ordinary skill in the art to embody and implement the systems and processes herein described. It is important to understand that embodiments can be provided in many alternate forms and should not be construed as limited to the examples set forth herein. Accordingly, while embodiments can be modified in various ways and take on various alternative forms, specific embodiments thereof are shown in the drawings and described in detail below as examples. There is no intent to limit to the particular forms disclosed. On the contrary, all modifications, equivalents, and alternatives falling within the scope of the appended claims should be included. Elements of the example embodiments are consistently denoted by the same reference numerals throughout the drawings and detailed description where appropriate.

The terminology used herein to describe embodiments is not intended to limit the scope. The articles "a," "an," and "the" are singular in that they have a single referent, however the use of the singular form in the present document should not preclude the presence of more than one referent. In other words, elements referred to in the singular can number one or more, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises," "comprising," "includes," and/or "including," when used herein, specify the presence of stated features, items, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, items, steps, operations, elements, components, and/or groups thereof.

Unless otherwise defined, all terms (including technical and scientific terms) used herein are to be interpreted as is customary in the art. It will be further understood that terms in common usage should also be interpreted as is customary in the relevant art and not in an idealized or overly formal sense unless expressly so defined herein.

Biological validation developments relating to XF-pacing assays formed in accordance with the present invention include the following.

The XF data presented in Figure 2 show that the respiratory activity of L6 myotubes increases when cells are stimulated electrically during the assay. This increase is frequency-dependent and respiration indeed decreases again when electrical stimulation is ended. Acidification of the external medium reflects the respiratory increases. Increased respiration suggests that mitochondrial ATP synthesis through oxidative phosphorylation rises in L6 myotubes following electrical stimulation, whilst the additional medium acidification suggests ATP synthesis through anaerobic glycolysis increases as well. The combined total boost in ATP synthesis is an expected response to the increased energy demand from the contractions that are provoked by electrical stimulation. The traces shown in Fig. 2 were obtained from measuring the effect of electrical stimulation in a single XF24 well. A I OV-stimulation was applied at I Hz from time point A and the waveform frequency was increased to 5 Hz at point B. Stimulation was switched off at point C. Notably, these data demonstrate that cells may be stimulated electrically ( 10V and 20mA per pulse) using our assay for at least I hour without cell damage. In some embodiments the frequency and amplitude range of the applied electrical signal is extended. Specifically, it can be determined how contractile workload - modulated by electrical stimulation - influences oxidative and glycolytic ATP synthesis rates and how it alters allocation of the formed ATP to energy-demanding processes. These ATP fluxes can be calculated from XF data as recently described in detail by others [3]. The same XF data can reveal if and how skeletal muscle workload affects coupling efficiency of oxidative phosphorylation (cf. Fig. I ). The present invention also relates to the relation between skeletal muscle workload and nutrient supply, with emphasis on the fuel excess that causes insulin resistance [9]. L6 myotubes may be grown at a range of glucose concentrations between 5 and 25mM and be exposed to different combinations of saturated and unsaturated fatty acids including palmitate, stearate, linoleate and palmitoleate using protocols reported by us before [6].

The XF-pacing assay formed in accordance with the present invention can provide considerable benefits for both basic and clinical research. Electrical stimulation of skeletal muscle cells allows functional measurement of muscle energy metabolism under contractile workloads that are physiologically relevant. Such measurements will be crucial for establishing causality of mitochondrial involvement in obesity-related insulin resistance [6, 1 0], a feature of the Metabolic Syndrome that is largely responsible for the seemingly unstoppable type 2 diabetes pandemic [ 1 0]. An XF- pacing assay will also set the scene for mechanistic studies into the role of mitochondrial dysfunction in ageing-related (sarcopenia) and disease-related (cachexia) loss of skeletal muscle mass and strength [ I I ]. Related, measuring bioenergetics of contracting muscle in vitro benefits mechanistic studies in the fields of exercise, sports and rehabilitation science. Moreover, pacing cardiac muscle cells in XF analysers may offer a sensitive cardiotoxicity assay under workload conditions the heart encounters in vivo. Such an assay will benefit drug discovery eliminating those with undesirable side effects on cardiac function at an early stage during drug development. Notably, assays conducted using the present invention could lower the dependence of medical research on animal work as it allows in vitro bioenergetics assays under physiologically relevant conditions. As such, it offers ethical as well as cost benefits, and because of a relatively high experimental throughput, the invention will be of value in drug discovery programmes of the pharmaceutical industry. Seahorse maintains a database of published research applying their technology. This database of publications can be used to estimate the potential market size for our invention. As of June 201 7, 356/4275 publications (8.3%) involve muscle, 1 57 of which (3.7%) focus on cardiac muscle, and our invention will be of interest to all research groups behind this work. Moreover, a cellular bioenergetics assay that enables in vitro muscle screens under physiological workloads could generate a new market replacing animal-based work with our comparably inexpensive and high-throughput technology.

The technology could be adapted to become a stand-alone product that may be used in conjunction with Seahorse XF analysers as well as conventional microplate readers.

Technical description

Down-scaling existing electrical stimulation protocols to a 24-well plate format:

The Seahorse XF24 plates have 24 wells with a diameter of 5mm (cell growth area of 2cm ² ) and an effective assay volume of just 7μΙ, which poses a challenge in terms of electrical stimulation. Such stimulation is further complicated by fluorescent probes blocking access from the top of the wells. The present invention allows stimulation on this relatively small scale, and would indeed permit further down-scaling to 96-well XF96 plates.

Electrical waveform optimisation:

Ideal stimulatory electrical waveforms (i) provide sufficient electrical energy to depolarise the plasma membrane transiently, i.e., in a way that mirrors in vivo stimulation by nerves, and (ii) stimulate contraction without providing excessive energy that damages the plasma membrane and thus limits the cells' contractile ability. Stimulatory waveforms were extensively tested through microscopic inspection of the contracting cells. A 1 0-50ms bipolar pulse (negative first) applied at 1 0V with a 20mA current was found to be optimum. The contractile frequency was restricted to 5Hz (5 contractions per second), which allowed full contraction - above 5 Hz, the cells enter a tetanic state akin to cramp. Putting things in perspective, close to its capacity, a heart beats at 220bpm, i.e., 3.7Hz. Waveform voltage, pulse and frequency are computer- controlled. Electrical field distribution:

Cells were found to contract optimally when aligned in parallel to the applied electrical field across the maximum width of the well. Electrodes were designed accordingly, but further developments are envisaged, possibly with additional electrodes providing fields in other directions. Besides application in XF analysers, such a design could provide an electrical field during small-scale growth in a tissue culture incubator. Using lonOptix C-PACE technology, we found that cells indeed align to electrical fields during growth. Electrical circuit design to amplify electrical waveforms:

Wells containing cells and assay medium have a low and complex electrical impedance, which means that (the power required for) electrical amplification of signals applied to 24-well plates will readily distort the waveform. Such distortion was found to polarise the assay medium and thus cause cell-damaging electrolysis. Empirically, an H-bridge motor control circuit design was found to minimise this issue. This type of circuit is used in robotics to control fine motor movements. Three dual H-bridges (Fig. 3) allow independent electrical stimulation of 6x4 wells thus allowing 6 contractile regimes to be investigated during a single XF run. The H-bridges typically provide a maximum current of 2A when an external power supply unit is used to power the H-bridges (+ 5V), to generate the stimulatory signal (+/- 12V), and to provide GND. We envisage that a 24- or 96-H-bridge design will offer control in individual wells of XF24 and XF96 plates, respectively. The H-bridge-based signal generator may be accommodated within the XF24 analyser adjacent to the XF plate/cartridge. The micro-controller is programmable as to timing and frequency of electrical stimulation. Custom-written computer code may be used to enable instructions to be given from mobile and static devices. The code may be incorporated in existing software that controls the XF24 analyser or any other plate reader.

Electrodes:

Various conductive materials were tested and carbon fibre was found to offer the best solution to create the ideal surface area and electrode shape. For one embodiment the carbon fibre was shaped and set in PDMS silicone (e.g. Slygard 184) to the contours of the XF24 wells. Additional electrode materials that were explored include graphene, platinum and carbon nanotubes cast in PDMS. In some embodiments resins such as epoxy were used to encase the electrodes.

Carbon electrodes were empirically found to work well when 5mm wide, I mm thick, curved to the well shape, and when applied within the well in couples spaced 5mm apart. These electrodes have to be located at the base of the well and need a conductive surface I mm high in order to efficiently distribute the electrical field across the cells. The setup produces an electrical field of 2V/mm across the cells - stronger fields injured the cells and detached them from the base of the plate thus limiting contractile lifetime.

In some embodiments the electrodes are inserted immediately before the XF24 assay. In other embodiments the electrodes may be incorporated into the base of the wells allowing the easy transfer and connection of the cell plate and the ability to stimulate the cells during growth. Bespoke circuit boards can be mounted onto XF plates allowing simultaneous application of the electrical signal to all 24 wells (Figure 4). The circuit board can be made from (i) glass fibre with etched copper tracks (e.g. FR4), (ii) Kapton with either platinum- or gold-coated electrodes, or (iii) a PDMS system with imbedded flexible electrodes. The circuit board carrying the electrodes could also be affixed to the top of the XF24 microplate.

Advantages over existing products or processes

The present invention allows the electrical stimulation of contractile cells in XF microplates. Importantly, this means that skeletal and cardiac muscle cells can be studied under contractile workloads that mimic the physiological conditions that these cells face in vivo. The effect of specific contractile workloads on cellular bioenergetics can thus be quantified with our invention. Moreover, bioenergetic changes provoked by disease states and/or pharmaceutical agents can be probed in muscle cell models experiencing physiological workloads.

According to a further aspect of the present invention there is provided a protocol for growing skeletal muscle cells in a cell culture plate, comprising the steps of: providing a standard cell-matched culture medium; providing a plasma-treated cell culture plate; seeding the plate with actively growing myoblasts suspended in the cell culture medium; incubating the cells in an initial incubation step; filling the plate with further cell culture medium; incubating the cells in an intermediate incubation step for a period until the myoblasts stop dividing; replacing the cell culture medium with a reduced serum medium; incubating the cells in a further incubation step, periodically replacing the reduced-serum media to promote the formation of myotubes.

In some embodiments the cells are seeded at a concentration required to achieve approximately 10,000 cells/cm ² of the plate growth surface area.

The present invention provides a protocol to grow contractile skeletal muscle within microplates. This allows, for example, for the measurement of the bioenergetic behaviour of electrically stimulated skeletal muscle cells.

A reliable protocol has been developed to grow skeletal muscle cells within XF24 cell culture plates where the majority of cells are located under the fluorescent probes (other types of probes are, of course, possible with the general aim being to gather extracellular flux data) and achieve a state of differentiation that enables contraction.

Some aspects and embodiments of the present invention require some/all of:

I ) The cells are not completely attached longitudinally to the bottom of the plate, allowing them to shorten during contraction.

2) The cells are not coated significantly by extracellular protein that, from our observations, insulates cells from the electrical current.

3) The cells are seeded into the centre of the well in a 5-20μΙ spot and are left to attach for 45 min before being covered in approximately 500μΙ of growth medium. In some embodiments the cells are left for up to I hour, but never longer to prevent cells from drying out. In some embodiments the protocol is optimised for Seahorse extracellular flux (XF) analysers or standard microplate readers, and the electrical stimulation of contractions within these machines. An embodiment of the present invention providing a cell culture plate stimulation system is shown in Figure 5.

An embodiment of the control circuits contained in B is shown in Figure 3. In this embodiment components A, B, C and D comprise the invention.

In this embodiment components E, F and G are part of existing microplate readers such as the Agilent XF24. C can be used to upload a preset program before the analysis or be permanently connected to the control circuit, providing real-time control.

An embodiment of the D Electrodes, circuit layout is shown in Figure 4. Component D is fitted into E to provide a single component.

D can be fitted either to the top or bottom of the microplate E.

Typical data outputs (G) are shown in Figures I and 2.

In this embodiment a microplate is provided with electrodes. In this embodiment the control circuit, containing the H-bridges is not part of the microplate and electrodes.

The PCB controller is separate and connected by a set of wires.

In this embodiment the PCB controller system is next to and connected to the microplate inside the machine. In this embodiment the PCB controller sits inside the machine next to the microplate. In other embodiments, however, it could be moved outside the analyser utilising a longer set of connection wires. The microplate is plugged into a connector located inside the analyser on a tray that receives the microplate.

In this embodiment the analyser collects data from the microplate using its standard built in fluorescent probes. It is blind to the presence of the stimulator system and does not receive any data from it.

Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiments shown and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.

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