text:
Gerald A.
Laboratory for Sensors, IMTEK – Department of Microsystems Engineering, University of Freiburg, Germany. E-mail: email@example.com; Tel: +49 761 203 7265
First published on 5th April 2018
Microsensor systems for cell metabolism are essential tools for investigation and standardization in cell culture. Electrochemical and optical read-out schemes dominate, which enable the marker-free, continuous, online recording of transient effects and deliver information beyond microscopy and end-point tests. There has been much progress in microfluidics and microsensors, but the translation of both into standard cell culture procedures is still limited. Within this critical review, we discuss different cell culture formats ranging from standard culture vessels to dedicated microfluidic platforms. Key aspects are the appropriate supply of cells, mass transport of metabolites to the sensors and generation of stimuli. Microfluidics enable the transition from static to dynamic conditions in culture and measurement. We illustrate the parameters oxygen (respiration), pH (acidification), glucose and lactate (energy metabolism) as well as short-lived reactive species (ROS/RNS) from the perspective of microsensor integration in 2D and 3D cell culture. We discuss different sensor principles and types, along with their limitations, microfabrication technologies and materials. The state-of-the-art of microsensor platforms for cell culture is discussed with respect to sensor performance, the number of parameters and timescale of application. That includes the advances from 2D culture to the increasingly important 3D approaches, with specific requirements for organotypic microtissues, spheroids and solid matrix cultures. We conclude on the current progress, potential, benefits and limitations of cell culture monitoring systems from monolayer culture to organ-on-chip systems.
Jochen Kieninger
Dr.-Ing. Jochen Kieninger studied Microsystems Engineering at the University of Freiburg. In 2003, he received his diploma in engineering. Afterwards, he worked in the Laboratory for Sensors at the Department of Microsystems Engineering (IMTEK) and in the School of Soft Matter Research at the Freiburg Institute for Advanced Studies (FRIAS). In 2011, he completed his PhD on "Electrochemical microsensor system for cell culture monitoring". In 2012, he was nominated as a lecturer and has since been working as a senior scientist in the Laboratory for Sensors. His research interests are electrochemical sensors, biosensors, microsensors for neurotechnology, cell culture monitoring, electrochemical methods for MEMS and microfabrication.
Andreas Weltin
Dr.-Ing. Andreas Weltin received his diploma (2008) and doctoral (2015) degrees in Microsystems Engineering from the University of Freiburg. Since 2015, he has been a group leader at the Laboratory for Sensors. In 2016, he received the nationwide 2nd Klee prize from DGBMT for his dissertation on in vivo sensors. He has developed and applied numerous microsensor platforms for 2D/3D cell culture metabolism. His research interests include the fabrication of (bio-)analytical microsystems, bio- and chemo-sensors, electrochemistry, microfluidics, and biomedical in vitro and in vivo applications in cancer research and neuroscience (presently, e.g., organ-on-chip systems in cancer therapy, characterization of neural interfaces).
Hubert Flamm
Dr.-Ing. Hubert Flamm studied Microsystems Engineering and obtained his diploma degree from the University of Freiburg in 2009. From 2009 to 2017, he worked in the Laboratory for Sensors at the Department of Microsystems Engineering (IMTEK). In 2014, he received his doctoral degree studying about "Electrochemical Microsensors for Superoxide Monitoring in Cell Culture". His research focuses on the development of new micro-sensor platforms for the measurement of short-lived reactive oxygen and reactive nitrogen species in 2D cancer cell research and the application of microtechnology, electrochemical methods and microfluidics in analytical microsystems and chemo-sensors.
Gerald A. Urban
Prof. Dr. Gerald A. Urban received his Diploma in Physics at the Technical University Vienna. Afterwards, he became a research assistant at the Neurosurgical Department of University Hospital Vienna. He received his PhD in Electrical Engineering, was a co-founder of the company OSC, Cleveland, and became a scientific director of the Ludwig Boltzmann Institute for Biomedical Microengineering. In 1997, he became a full professor for Sensors at the University of Freiburg. He was a Dean and spokesman of the Academic Senate. He is a member of the Austrian Academy of Sciences and series editor of the SPRINGER series "Bio- and Chemosensors". His main research interest focuses on micro- and nanosensor applications.
In order to obtain relevant information from such in vitro models, it is important to acquire the actual state of the cells. Optical, continuous observation of cell morphology provides only basic information about the cellular state. Staining methods reveal more and also intracellular details. Typically, staining interferes with the cells, often needs fixation and thus can only provide end-point data. To address the cellular metabolism itself, observation of the living cells is needed, which is preferably carried out by accessing the small molecules involved in the metabolism (oxygen, glucose, lactate etc.) with microsensors. The usage of such sensors allows the continuous recording of transient mechanisms, which is often called metabolic monitoring. This is especially helpful in scenarios in which recovery or cyclic effects could be overseen if only end-point data is used.
Research using in vitro models along with the ability of metabolic monitoring can be driven by many different motivations. In fundamental research, the knowledge of metabolic functions can be of primary interest. In other fields, such as the study of gene expression or drug response, the metabolic state often determines the results, and thus, monitoring of metabolic parameters essentially contributes to the standardization of cell culture experiments.
Pharmacodynamics studies, drug testing and compound screening are all applications in which the metabolic monitoring provides indicating parameters for specific pharmacological interactions. Results from in vitro cultivation of patient material in personalized medicine can benefit from microsensor readings, as within the very limited time frame of typical clinical scenarios a large quantity of information can be obtained. In all these different fields of application, the in vitro models in combination with microfluidics and sensors provide a rather simple possibility to parallelize the experiments in a reproducible manner. This allows both a detailed parameter study and collection of sufficient data for good statistics. Many systems claim to provide easy scale-up for parallelization. However, it is rarely shown, and in most cases it would be technically challenging.
In our opinion, the available tools benefit a lot from great progress in microfluidic systems, while the application of microsensors therein still lags behind. Critically, this is particularly true for 3D cell cultures, although several promising approaches have been proposed.
Within this critical review, we discuss the different sensor principles for the most relevant metabolic parameters as well as microfabrication technologies and materials with respect to their applicability in mammalian cell culture monitoring systems. Then, we review the different systems described in the literature and commercial products. The key aspects of those systems are:
• How are the cells cultured?
• How do the metabolites come to the microsensors?
• Whether and how are stimuli generated?
Monitoring systems can be separated into dynamic systems comprising microfluidics and static systems with stagnant medium following the routine procedure in cell culture. Depending on the field of application, either the culture system should provide results very fast, or microsensors should be able to measure for longer time, possibly without the need for recalibration. Another important aspect is the number of different sensing parameters provided by the system. In Fig. 1, we summarize the needs for the different fields of application. The fields are clustered depending on the desired measurement duration and on the number of measured parameters from single to multiparameter monitoring. Drug screening systems should be optimized for acquisition of results within minimal time, while standardization of cell culture typically addresses many cell cycles resulting in demands for stable sensors over weeks.
|Fig. 1 Classification of cell culture monitoring systems by the needs of different fields of application.
Fig. 1 describes both needs which are essential in order to obtain meaningful results and needs describing the optimal case. This is especially true for the field of cell therapy. While successful measurements are possible with less capable systems, we think that cell therapy will be the field with the highest demand for long-term stability and number of parameters in the future.
We do not comprehensively treat metabolic monitoring in bioreactors because of its different environment for sensors, resulting in completely different aspects for size, integration, stability and functionality. However, some works describing microsensors dedicated to bioreactors are applicable to mammalian cell culture and organ-on-chip systems as well and were therefore included. An overview of selected cell culture monitoring systems is given in Table 1.
|Cell culture type
|Sensing principle
|Notable features
|2D, dynamic
|Amperometric (Pt)
|109, 110
|Glucose, lactate
|Amperometric biosensor (Pt)
|Sensing Cell Culture Flask
|2D, static
|Amperometric (Pt)
|Standard culture vessel
|28, 107
|Potentiometric (iridium oxide)
|Glucose, lactate
|Amperometric biosensor (Pt)
|Amperometric (Au)
|Differential pulse voltammetry
|Boero et al.
|2D, static
|Glucose, lactate
|Amperometric biosensor (Au)
|External biosensors
|Presens OxoDish
|2D, static
|Commercial, standard format
|Seahorse Bioscience
|2D/3D, static/dynamic
|Oxygen, pH
|Commercial, standard format, dip-in approach
|Bionas SC1000
|2D, dynamic
|56, 115
|Amperometric (Pd)
|Impedance (IDES)
|2D, dynamic
|Downstream biosensors on-chip
|Potentiometric (iridium oxide)
|Glucose, lactate
|Amperometric biosensor
|Misun et al.
|3D spheroid, dynamic
|Glucose, lactate
|Amperometric biosensor (Pt)
|Hanging-drop network, in situ biosensors
|Bavli et al.
|3D spheroid, dynamic
|Luminescence (tissue embedded)
|Commercial, external downstream biosensors
|Glucose, lactate
|Amperometric biosensor
|Zhang et al.
|3D spheroid, dynamic
|Optical absorption
|Multiple sensor units, fluidic breadboard
|Cyclovoltammetric (Au)
|Weltin et al.
|3D spheroid, static
|Amperometric (Pt)
|Standard culture vessel, in situ biosensors, dip-in approach
|Amperometric biosensor (Pt)
|Domansky et al.
|3D, scaffold-based, dynamic
|On-chip pumps, dip-in approach
Keeping in mind that this is a severe abstraction, we focus on the simplified energy metabolism of glucose as illustrated in Fig. 2 for the discussion of the cell culture monitoring systems. In the first step, glycolysis, glucose is broken down into pyruvate molecules. Depending on oxygenation around the cell, pyruvate is further catabolized following an aerobic or an anaerobic pathway. Because of its overall higher efficiency (the amount of energy generated per glucose molecule), the aerobic pathway is preferred.
|Fig. 2 Simplified energy metabolism of glucose illustrated at the location where it takes place in a cell. Based on the availability of oxygen, an aerobic or an anaerobic pathway is possible. The substances highlighted in green can be observed in the extracellular space and are potential candidates for cell culture monitoring using microsensors.
In the cytosol, glycolysis occurs, in which the six-carbon sugar glucose is broken down into two three-carbon sugars and converted further into two pyruvate ions. In the case of sufficient oxygen, pyruvate is oxidized within the mitochondria to acetyl-CoA releasing CO2. Acetyl-CoA is the starting point of the citric acid cycle, in which further energy is generated, with CO2 and protons as by-products. Downstream of the citric acid cycle, in the oxidative phosphorylation, further energy is harvested by reduction of oxygen to water. If oxygenation is not sufficient, oxidative phosphorylation cannot occur, and the citric acid cycle stops, resulting in accumulation of pyruvate ions in the cytosol. In this case, the anaerobic pathway, the fermentation of pyruvate into lactate and release of a proton, sets in.
Cell culture monitoring using microsensors per se can measure extracellular substances only. Except for cultivating isolated mitochondria, only the substances marked green in Fig. 2 can be accessed directly. The individual steps of the energy metabolism of glucose can only be seen indirectly.
Glucose as the major energy source in cell culture is available in the cell culture medium in high concentrations up to 10 mM or more. Physiological levels are typically lower (few mM), but glucose is not the sole energy source in vivo. In cell culture, glucose levels decrease depending on medium volume, cell density and metabolic activity and often reach values below the mM range before the medium is exchanged.
The concentration of dissolved oxygen is determinant for many different biological processes.3 Aerobic cell culture conditions (e.g. 5% CO2, 95% air) result in dissolved oxygen concentrations in the range of 200 μM. Depending on the type of culture, pericellular values are much lower because of the formation of diffusion gradients (see Fig. 3A). Typical oxygen concentrations in healthy tissue are between 20 and 150 μM. Pathologically low oxygen conditions (hypoxia) can be found, depending on the origin of the cells, at 25 μM or lower.4 In cell culture, it is important to establish a well-defined pericellular oxygen concentration in order to ensure the reproducibility of the desired cellular state. In situations with limited medium volume or the possibility of formation of gradients, cellular respiration rates can be accessed. Typical rates are in the range of 200 fmol h−1 per cell (T-47D breast cancer cell line).5 The aspect of oxygenation and oxygen control in cell culture has been discussed recently in more detail in this journal.6,7
|Fig. 3 Concentration gradients in static 2D (A) and 3D cell culture (B), as well as dynamic cell culture monitoring with active transport (C). A: A monolayer of cells grows on the bottom of the culture vessel. A concentration gradient along the medium height occurs due to cellular production or consumption of substances. B: 3D cell agglomerates grow, often heterogeneously, in a 3D matrix or in medium. A radial concentration gradient occurs around the microtissues. Typically, there is an even stronger gradient inside the microtissues. C: A monolayer of cells grows inside a microfluidic microsensor system (microphysiometer). The small volume in the microchannel needs to be exchanged periodically using a pump to ensure cell survival. Large concentration changes of metabolites due to the small medium volume can be measured in situ, or metabolites are transported to downstream sensors by microfluidics.
Lactate ions are the end product of the anaerobic pathway. Initially, the cell culture medium contains no or, in case serum was used to prepare the medium, up to a few mM lactate. Under typical culture conditions, lactate concentrations can rise up to few mM, usually not higher than half of the initial glucose concentration in the medium. Taking into account the limitations of the simplified model described in Fig. 2, the fraction of twice the number of lactate molecules produced by the number of glucose molecules consumed can be used as a measure for the fraction of the anaerobic pathway. In a more detailed view, it should be considered that, depending on the cell type, lactate can be used as an energy source or even metabolized to glucose, such as when liver cells convert lactate produced in the muscles back to glucose (Cori cycle).
Dissolved CO2 gas and protons (pH) are interlinked by the dissolution of CO2 and formation of bicarbonate ions (eqn (1)). Therefore, monitoring of pH is often preferred over measuring dissolved CO2 gas in cell culture monitoring applications.
Additionally, the pH in a typical cell culture medium is stabilized by sodium bicarbonate and cultivating the cells in an incubator atmosphere containing CO2 gas (e.g. 5%), which results in a strong buffer system following eqn (1). The pH of the typical cell culture medium is adjusted to 7.4, decreasing over time due to cell metabolism to values not much below pH 7 under appropriate culture conditions. In case cellular acidification should be accessed and measured as acidification rates with microsensors, a small total medium volume along with a weakly buffered medium is required.
For parameters like glucose, the medium acts as a finite source; the cells are the sink. In the case of lactate and other waste products, the only source is the cells. For those parameters with a finite source or with just cells as the source, measurements at any position along the medium height of the 2D cell culture provide qualitatively meaningful results. However, to obtain quantitatively correct readings, a defined sensor position next to the cells is necessary. In contrast, for parameters like oxygen, the cells act as a sink, and the top surface of the medium (the incubator atmosphere) is an infinite supply. The established gradients and therefore the oxygen concentration next to the cells (pericellular) depend on the aerobic cell metabolism, which may be a function of the pericellular oxygen concentration itself.5 In this situation, it is essential to have sensors next to the cells in order to obtain meaningful sensor readings.
Three-dimensional (3D) cell cultures gain popularity because they resemble the in vivo situation more closely and show more organotypic properties than 2D cultures.8–10 Such properties make them promising models for drug and toxicity screening, where microphysiometry has been applied in 2D to measure cell metabolism, and also in tissue engineering, cancer research and multi-organ modeling. In 3D culture, cells are typically either mutually adherent but non-substrate adherent aggregates,11e.g. spheroids,11,12 in a scaffold-free medium or mutually adherent and embedded within a gel/solid matrix, such as Matrigel13 or other scaffolds. Today, spheroids are one of the most used 3D cell culture models, also in microfluidic systems. They can be formed in different ways which include:11,12 aggregation of cells by gravity in hanging droplets at the air/liquid interface; use of vessels, reactors or labware with non-adhesive/cell-repellent surfaces and shaking, spinning, rotating or centrifuging such vessels; employing microwells or molds made from non-adhesive materials; growth within or around a specific matrix/hydrogel, bead or emulsified droplet. The typical spheroid size is in the range of several hundred micrometers. Gradients for metabolic reagents and products can be found in the matrix outside the spheroid. Additionally, and usually dominant, there are gradients which occur within the spheroids themselves, as illustrated in Fig. 3B. Oxygen, for example, depletes along the radius towards the center. Assuming appropriate culture conditions, it is possible to achieve neighboring layers with normoxic and hypoxic states, which are an essential model system in tumor research.14 Spheroids can be understood as an inverse model of the tumor geometry: in tissue, oxygen and nutrients' concentrations drop radially along the distance from a blood vessel in the center, whereas in a spheroid, the lowest oxygen and nutrients' concentrations can be found in its center. Especially for the method of hanging droplets, microfluidic systems have been applied to enable parallelization and enhance reproducibility.15–17 Access to metabolic parameters using microsensors is often limited to the medium surrounding the spheroid.18,19 Measurement of values or even tracing of gradients inside the spheroids can hardly be achieved by microsensors as typical sensor geometries are in the same size as the diameter. Although the penetration of the spheroid with sensors in the format of a microneedle is feasible,20–22 the results would strongly be influenced by the physical presence of the sensor.
Other 3D cell culture formats comprise cells growing equally distributed in a 3D cell culture matrix in a tissue-like manner, sometimes also comprising vessel-like structures to mimic vascularization.23,24 In such systems, sensors can be used to provide concentration readings at distinct points, e.g. outside the matrix and in the middle.
Microsensors can also be used to monitor suspension cell culture, however these systems typically relate to the field of bioreactors, which is not covered by this review.
|Sensor types
|Relevant range in cell culture
|Analyte consumption
|Direct amperometric sensor
|0–200 μM (0–20 kPa)
|Clark-type sensor
|Depends on the principle
|Optical sensor (luminescence/fluorescence)
|Potentiometric sensor
|Optical sensor (luminescence/fluorescence)
|Electrochemical biosensor
|0–15 mM
|Optical biosensor
|Electrochemical biosensor
|0–25 mM
|Optical biosensor
Electrochemical oxygen sensors are based on the reduction of molecular oxygen at a noble metal electrode (usually platinum). Depending on whether the electrode is directly in the cell culture medium, those sensors are called direct amperometric sensors or, in case there is an independent sensor electrolyte separated by a gas-permeable membrane, they are called Clark-type sensors. It has to be noted that sometimes in the literature the term "Clark-type" is used for all types of electrochemical oxygen sensors. The advantage of the Clark-type arrangement is the independence of the electrode reactions from interfering or electrode poisoning substances from the cell culture medium, but at the cost of higher technological effort.25–27 Application of chronoamperometric protocols comprising cleaning steps enables stable measurements in cell culture medium including serum also with direct amperometric sensors.28 For both types of amperometric oxygen sensors, the transfer function is linear assuming sufficient diffusion limitation within the sensor membrane. Typically, the offset of such sensors is close to zero allowing one-point calibration at the beginning of the cell culture experiment, which suits the conventional cell culture routine much better than all other sensing methods requiring at least two different oxygen concentrations during the calibration procedure. A variant of the amperometric oxygen sensor is to use the local pH change caused by the oxygen reduction process to generate the measurement signal using pH-sensitive field effect transistors.29,30 A major advantage of this approach is the possibility to use the same readout circuitry as that used for pH sensing.
Potentiometric sensors with their inherent logarithmic transfer function (following the Nernst equation) would often fit better to the possible oxygen concentration ranges.3 However, to our knowledge, no potentiometric oxygen sensor has been applied successfully to monitor dissolved oxygen concentration in cell culture.
Optical oxygen sensors are based on fluorescence quenching in an appropriate dye by molecular oxygen.31 Measurement principles include the evaluation of fluorescence amplitude, fluorescence lifetime or phase shift in the case of modulated excitation light. For all principles, the transfer function follows the non-linear Stern–Volmer relation. This relation states that the output of the sensors is inversely proportional to the concentration plus a constant. This means that there is no clear zero-point and therefore two-point calibration is needed. The fluorescent dye can be embedded in a polymer as a single sensor spot or spread in a membrane coating on the whole surface of the cell culture vessel in order to measure spatial changes in oxygen concentration.32,33 In both cases, fluorescence excitation and readout can be carried out directly using LEDs or coupled through an optical fiber outside transparent cell culture vessels. This is a big advantage with respect to easy handling and sterilization of the cell culture vessels and allows separation of the single-use sensor spot from the reusable optical setup. Similar sensor spots can be read out with an inverted microscope.34
Potentiometric pH sensors based on metal oxides, such as iridium oxide,42–46 tungsten oxide47–49 or ruthenium oxide,50–52 can also be used on transparent substrates and are often preferred because of their simple integration. However, the overall accuracy is also dependent on the stability of the reference electrode. Optical pH sensors are based on fluorescence quenching at alkaline pH, but usually need, in contrast to optical oxygen sensors, a second reference dye (dual lifetime referencing).53
LAPSs, ISFETs and potentiometric sensors have a logarithmic transfer function referring to the proton concentration (i.e. linear with pH), while optical pH sensors follow a more complex non-linear function.54 Both transfer functions are well suited for larger pH changes. The case of typical changes within cell culture of less than one pH unit requires very stable sensor signals over days. In order to stimulate faster changes as response to a stimulus, a medium with low buffer capacity can be used.55–57
The physiological concentration of NO is in the range of only pM to μM during burst events.61,62 NO radicals show a typical diffusion of around 150–300 μm within a 4–15 s range in biological media,63–67 whereas other sources report a diffusion of 10 μm within 15 ms.68
Under physiological conditions, the half-life of free superoxide radicals is very limited in the range of milliseconds to some few seconds. This short lifetime results in a low cellular basal concentration of some pM (ref. 69) and a free diffusion path length of around 40 μm under in vivo conditions.70 The spontaneous decomposition rate of superoxide is strongly dependent on the local pH.
The short half-life and diffusion length of ROS and RNS arise from spontaneous or enzymatic decomposition and high reactivity with a bundle of different biomolecules, including membrane lipids, proteins, nucleic acids, other reactive species and transition metal ions. One example for a fast conversion is the reaction of NO* with O2*− to ONOO− with an almost diffusion-controlled rate (the rate constant is 4–16 × 109 mol−1 L s−1).71 As a consequence, the positioning of a sensor in the diffusion zone of ROS/RNS and the cellular microenvironment has tremendous influence on the signal height,66e.g. a complete signal loss at a position 1 mm away from single cell for NO measurement.68 Also, Brovkovych et al. showed the direct influence of sensor tip distance on the NO signal in a single endothelial cell experiment.72 Lee et al. demonstrated the impact of electrode and sealing size at a needle-shaped NO microsensor setup.65 It becomes obvious that a closer electrode cell distance (artificial synapse arrangement) increases the collection efficiency of the sensor. On the other hand, the diffusion of potential reaction partners like oxygen or arginine may be hindered by the sensor body, which leads to analyte accumulation.
In particular, in planar, chip-based sensor approaches with randomly growing cells, the complex hemispherical diffusion profiles for ROS and RNS can hardly be predicted. Further, the relationship between the diffusion profile/electrode overlap and electrode size may lead to a misinterpretation of the measured extracellular concentration when compared to an external calibration setup with homogeneously distributed analyte standards, even with correct sensor readings.
Microfabrication techniques allow for sensor design to measure within the expected area of occurrence and enable, along with appropriate electrochemical techniques, label-free measurement with sufficient spatial and time resolution. One approach to address the low concentration profiles of short-lived species might be the utilization of ultra-microelectrode (UME) arrays. A higher mass transfer rate, compared to millimetric electrodes, in terms of studying fast kinetics, and a better signal/noise ratio are the main aspects advocating their use, whereas smaller overall signals from lower electrode areas challenge the readout electronics' performance. Zhang et al. described an array of 2500 ultra-microelectrodes (carbon, 2 μm diameter) exploiting the effect of UME arrays for NO measurement.73 This report demonstrated the occurrence of steady-state regimes and the additivity of electrode currents. Later, the electrodes were coated with permselective membranes (Nafion and a commercial membrane from World Precision Instruments, USA) to enhance the selectivity towards NO, which can be detected by amperometry at 0.86 V vs. Ag/AgCl.
Parallel measurement of both ROS and RNS in stimulated adherent cell culture under static conditions was successfully demonstrated by Chang et al.74 They used planar sputtered gold/cytochrome c electrodes for superoxide and screen-printed carbon electrodes with electrodeposited tetrasulfonated nickel phthalocyanine (NiTSPc) for nitric oxide detection. The system consists of a full 24-well format sensor array and makes use of standard transwell inserts for cell culture pre-cultivation. The transwell is inserted into the measurement setup and avoids direct electrode/cell contact by the transfer membrane.
Another interesting approach by Isik et al. is the use of pyramid-shaped Pt tip electrodes on chip, to avoid direct cell–electrode contact and possible cell death upon electrical polarization.75 The electrode tips are located between/above the cells, which enables a measurement of NO within its diffusion zone. The tips were modified with NiTSPc to improve their NO sensitivity and measurement with HUVEC cells was performed amperometrically at 0.75 V vs. Ag/AgCl. The study focused on adherent cell line cultivation under static conditions, but the 3D geometry of the electrodes might pave the way for measurement in tissue slices or 3D cell culture models.
Electrochemical measurement of ROS and RNS molecules makes use of specific redox potentials to discriminate between the main interfering substances, whereas the high oxidation potential especially for NO (>700 mV vs. Ag/AgCl) is not suitably specific. If high time resolution is required, fast-scan voltammetry and amperometric measurements are the methods of choice, but also with limited selectivity. Therefore, techniques like differential pulse voltammetry are a good compromise between acceptable time resolution and increased selectivity.
Alternative approaches to reduce cross-sensitivities use permselective membranes to cover the sensing electrodes, allowing only the analytes of interest to reach the electrode surface. Typical membrane materials in use are hydrogels, Nafion, polypyrrole, o-phenylenediamine or polymethylcellulose. In addition, we demonstrated selective and robust in vitro measurement of the superoxide radical by direct oxidation on a planar gold microelectrode covered with a polyethylenimine membrane.76
However, none of the described monitoring systems used integrated circuits to enhance signal performance or even eliminate external instrumentation. The sensor chip which is in direct contact with the cells should ideally be used once and therefore chips with integrated circuits generate unreasonably high costs. A critical disadvantage of silicon as a substrate is its opaqueness for visible light. Especially in the case of 2D cell culture, standard routines include optical inspection of the cells with through-light microscopy. Also, for 3D systems, optical access is beneficial. In the case of microfluidic systems, observation of bubbles simplifies successful measurements.
Approaches to introduce transparency by integration of a transparent window into the silicon chip79 or small silicon chips into glass substrates39 were mentioned. From today's perspective, the benefit of such approaches compared to the technological costs does not seem justifiable.
Glass substrates allow optical inspection of the cells as well as the possibility to use optical markers in the cell culture parallel to the microsensors. Thin-film processing for electrochemical sensors is possible using metals like platinum or gold along with silicon nitride or silicon oxide for passivation. Electrodes can be modified by electrodeposition of other metals onto the thin-film layers or coating with polymers similar to backend processes employed after CMOS fabrication in the case of silicon substrates.28,57
Polymer-based systems can be flexible, such as dip-in microsensors based on polyimide,18,83 or used as stiff materials comparable to other cell culture labware, such as well plates with integrated optical sensors.32 Gas permeability is excellent in silicones, which can be employed for gas perfusion into the cell culture. Polydimethylsiloxane (PDMS) replicas are often used to form channels on top of glass chips in order to combine sensor chips with microfluidics.
Sensor functionalization for optical sensors is performed by application of the fluorescent dye dissolved in a membrane material, typically made of silicone or similar gas-permeable polymeric material.
For sensor systems fabricated in CMOS technology, gamma sterilization is often not preferred because of altered characteristics mainly due to generation of defects in the gate oxide. Elevated temperature (autoclaving) is often avoided because mechanical stress can cause failure in hybrid mounted sensor chips. Biosensors comprising enzymes rule out higher temperature at all, while ethanol should not come into contact with the enzyme membranes. For biosensors, gamma sterilization is often a good choice if enzyme stability is considered.
The majority of monitoring systems focusing on oxygen as the sole parameter employ optical oxygen sensors based on fluorescence quenching (Fig. 4A). Those works cover toxicity studies,32 evaluation of oxygen consumption rates,89 investigation of hypoxia for cancer research90 or in non-tumor cells,91–93 optimization of culture conditions,94 stem cell differentiation,95 and endocrinology.96 Also, electrochemical oxygen sensors were used, mainly Clark-type27 or direct amperometric sensors.28 In our opinion, electrochemical sensors are only reasonable in the case of systems for multi-parameter monitoring, especially in combination with biosensors, or if very low oxygen concentrations need to be measured.
|Fig. 4 Sensor systems for metabolic monitoring in 2D static culture. A: Optical monitoring system to measure oxygen at the bottom of cell culture wells32 (reprinted with permission from Elsevier). B: Microdialysis system to monitor glucose and lactate102 (reprinted with permission from Elsevier). C: Multiparameter monitoring on a silicon chip with oxygen, pH, glucose and lactate sensors105 (reprinted from Pemberton et al., 2016, DOI 10.3390/s141120519, under CC BY 4.0). D: Sensing Cell Culture Flask: a transparent sensor chip with different electrochemical sensors is embedded in the bottom of a tissue culture flask.107
Instead of integrating oxygen sensors into the bottom of the cell culture area, an approach was described using integrated sensor strips enabling measurements at different heights above the bottom of the cell culture.83 This system is intended for determining oxygen gradients in stagnant medium above the cells in 2D culture, but would be an interesting approach to access oxygenation in 3D cell culture as well.
Another group of systems focuses on oxygen and pH as measurement parameters, for which mainly optical sensors are used.97,98 Further steps towards comprehensive multiparameter monitoring of cellular metabolism is the integration of biosensors into the sensor systems for static culture conditions. Systems were proposed using biosensors combined with microdialysis to measure glucose and lactate in static cell cultures (Fig. 4B).99–102 Glucose was also measured directly using a sensor chip dipped into a static cell culture.103,104
A multiparameter system measuring oxygen, pH, glucose and lactate using silicon chips was described for static cell culture in wells (Fig. 4C).105,106 The Sensing Cell Culture Flask (Fig. 4D) was introduced as an electrochemical sensor platform on transparent glass chips, embedded into the bottom of conventional tissue culture flasks, with sensors for oxygen, pH, glucose and lactate.28,76,83,107
The first microphysiometer (Fig. 5A) was based on a silicon chip with an integrated light-addressable potentiometric sensor (LAPS) for pH measurement.35,77 A sample of 104 to 106 cells was inserted onto a membrane. A plug with vertical in- and outflow channels was inserted for operation, reducing the chamber volume to 2.8 μL for measurement,108 and a peristaltic pump was connected. The flow was stopped for around 30 s, during which extracellular acidification changed the pH in the range of 0.1 units. The system was later commercialized as the Cytosensor36 and enhanced by other sensor types. By insertion of platinum wires into the sealing plug and subsequent polishing, microelectrodes were formed above the cell layer. Electrochemical biosensors for glucose and lactate and dissolved oxygen sensors were integrated.109–111 Newer implementations of the LAPS microphysiometry principle exist.37,38,112 With similar protocols, pH was also measured by means of ion-selective field effect transistors (ISFET) using CMOS technology on silicon chips.78,79,113 ISFETs also allowed the determination of dissolved oxygen by local pH changes.30 More common is the direct amperometric measurement of oxygen by reduction at noble metal electrodes.57,110,114
|Fig. 5 Sensor systems for metabolic monitoring in 2D dynamic culture (microphysiometers). Cells are cultured directly on microsensor chips. For measurement, the medium volume is reduced to a few microliters and periodically exchanged by microfluidics. A: The Cytosensor microphysiometer based on a silicon chip, in which pH is measured with a light-addressable potentiometric sensor (LAPS). Amperometric microsensors are included by integrating wires in the cover111 (reprinted with permission, Copyright 2012 American Chemical Society). B: Silicon-based system with planar sensor integration for pH (ISFET), oxygen (amperometric) and cellular adhesion (interdigital electrode)56,115 (reprinted with permission from Elsevier). C: Glass-based system for better microscopy with in situ amperometric oxygen sensors (platinum), potentiometric pH sensors (iridium oxide) and an interdigital electrode, as well as amperometric glucose and lactate biosensors integrated in the downstream microfluidics.57,107
Another parameter of interest in such systems is cellular adhesion, which is measured by impedance analysis.78,79 Attachment and spreading of cells on interdigital electrode structures (IDES) is determined by capacitance changes. Adhesion can be monitored continuously during all phases of cell growth, and is mostly independent of flow. The combination of IDES-based impedance monitoring with ISFET-based pH sensing and amperometric oxygen measurement for drug and toxicology screening on a silicon chip was commercialized by Bionas (Fig. 5B).56,115 The key feature of microphysiometers, in comparison to traditional end-point tests, is the rapid determination of metabolic rates in a continuous, online measurement (Fig. 6A), which reveals transient recovery effects of cellular metabolism and allows the investigation of pharmacodynamics in drug screening (Fig. 6B) and cancer research.56,57,116
|Fig. 6 Typical measurements from dynamic cell culture monitoring with microsensors. A: Extracellular acidification of 2D cells during the stop phase (pump off) in a microphysiometer system and return to the baseline during the flow phase (pump on)56 (reprinted with permission from Elsevier). B: Metabolic rates for acidification, respiration and adhesion extracted from such an experiment. Rates change upon substance exposure, and continuous, online monitoring allows the measurement of pharmacodynamics and recovery effects in the drug screening application56 (reprinted with permission from Elsevier). C: Biosensor measurement of lactate production from 3D microtissue spheroids in hanging droplets, including the blank signal, which shows lactate production and return to baseline depending on the flow profile19 (reprinted from Misun et al., 2016, DOI 10.1038/micronano.2016.22, under CC BY 4.0).
Particularly for enzyme-based biosensors which produce hydrogen peroxide as an intermediary product, sensor placement outside the cell culture chamber is desirable to reduce oxidative stress. We demonstrated the placement of glucose and lactate biosensors in the outlet channel (Fig. 5C).57,117 Undiluted medium from the cell chamber was transported to the biosensor array and measured during the flow phase. Amperometric oxygen sensors based on platinum electrodes and potentiometric pH sensors based on iridium oxide electrodes were included in the cell culture chamber. In contrast to silicon-based devices, we integrated these electrochemical, multiparameteric sensors on a transparent glass chip, which facilitates microscopy. Other groups focused on reduction of the volume to the sub-microliter range for glucose118 and lactate119 measurement or alternative fabrication methods such as screen- and inkjet-printing.120
The method and systems commercialized by Seahorse Bioscience fall between static and dynamic monitoring.55 Cells are cultured in largely standard microtiter plates, into which a sensor probe is lowered, temporarily reducing the volume above the cells to around 2 μL. Oxygen and pH are measured using optical sensors. Pneumatic reservoirs above the medium enable the addition of test compounds, and the movement of the sensor tip mixes the medium. Metabolic rate determination within minutes is possible, but options for microfluidics and sensor placement are limited. The system allows parallel experiments in the entire plate with automated sequential readout. From an engineering standpoint, using microphysiometer systems seems straightforward. However, their operation with a combination of fluidics, sensors and cell handling is more complex than standard procedures in cell biology. The amount of data acquired is high compared to end-point tests and requires at least some basic data processing to obtain meaningful results. Valuable efforts to automate operation121 and parameterize data processing122 have been made. However, few novel concepts or parameters have emerged within the last decade, and many existing systems have faded away. Other systems never left academia and might have suffered from reliability problems. Their complexity and their limited compatibility with true high-throughput screening are drawbacks which still persist even among the novel approaches for 3D cell cultures.
Many microfluidic systems have been developed for the formation, trapping and culture of non-adherent microtissues, but very few include sensors to measure cell metabolites. Systems are often microfluidic channel networks fabricated from molded or thermoformed polymers with small wells to trap the microtissues, while the medium flows over or alongside the well.123–127 Paper has also been used for both fluidic compartments and cell scaffold.128,129 Supernatants and perfusion media are then transferred to external readers or analysed in situ. Measurement is often limited to viability testing by optical methods such as fluorimetric live/dead staining.16,130 As these systems often require periodic or constant perfusion, a strict differentiation between dynamic and static measurement is more difficult.
The straightforward, static dip-in approach is to measure metabolites directly in the stagnant supernatant in standard culture vessels, i.e. microtiter plates. Here, two difficulties arise: due to the low cell number, concentration changes are small, and medium exchange happens only every few days. This requires highly sensitive and long-term stable sensors since the fluid volume cannot be reduced indefinitely. We showed the direct measurement of lactate and oxygen using electrochemical microsensors in standard 96-well plates containing a single microtissue spheroid.18,131 Needle-type sensor strips132 with microelectrodes at the tip were dipped into and remained in the well over days. The integration of the sensor system into the standardized setup enables compatibility with standard handling procedures. Lactate production rates from 2000-cell spheroids in the range of 5 μM h−1 were measured over up to three days. Oxygen measurement in the direct vicinity of the spheroids also revealed no hypoxic conditions in the well even though assumingly anaerobic metabolic pathways were active. By measuring altered metabolic rates under drug exposure, we demonstrated that metabolic access yields valuable information in drug screening, which complements and enhances microscopy or viability tests. Generally, electrochemical systems with electrode sizes in the micrometer range allow a highly localized measurement.
As in 2D systems, microfluidics can be utilized to reduce total media volumes and thus increase concentration changes. Collection and undiluted transport of media to downstream sensors enables the integral measurement over numerous microtissues. Such a system was realized for a multiwell bioreactor for hepatic spheroids (Fig. 7A).133 Nine microtissues were kept in approx. 1.2 μL wells each, within a gas-tight bioreactor with constant perfusion. Sensors included in situ oxygen sensors composed of tissue-embedded phosphorescent beads and commercial amperometric biosensors for glucose and lactate off-chip, in a combined outlet channel.134 Oxygen was measured continuously over 28 d and consumption rates were determined. After 4 d of adaptation, distinctly lower concentrations than air saturation were measured in the microwells. Glucose and lactate metabolism from all microtissues combined was measured from 40 μL medium extracted from the bioreactor. Microfluidic switching allowed frequent calibration of the biosensors with external solutions to account for drift of the sensors. A comparable concept for multi-organoid drug screening has been shown recently,135 in which cardiac and liver organoids within a microfluidic network were combined with optical pH and oxygen sensors,136 as well as electrochemical immunobiosensors.137 Even though these systems are designed to work largely automated, they require considerable technological effort and instrumentation to fabricate and operate both microfluidic networks and sensor periphery.
|Fig. 7 Sensor systems for metabolic monitoring in 3D cell culture. A: 3D microtissue spheroids trapped in microwells within a perfused microbioreactor. Oxygen and pH are measured optically through nanoparticles embedded in the microtissues. Glucose and lactate are measured using downstream amperometric biosensors connected by a microfluidic switchboard133 (copyright 2016 National Academy of Sciences). B: 3D microtissue spheroids cultured in interconnected hanging droplet networks. A glass-based sensor chip and microstructures allow fluidic connection between the drops and integration of in situ microsensors for, e.g. lactate, glucose or impedance19 (reprinted from Misun et al., 2016, DOI 10.1038/micronano.2016.22, under CC BY 4.0).
Hanging droplets are another method for scaffold-free culture of non-adherent microtissues. Cells agglomerate and form spheroids at the bottom of the hanging drop, driven by gravity and convection forces, where only a liquid/gas interface exists.16 By balancing inflow and outflow through active pumping17,138 or surface tension-driven flow,139 droplet size can be maintained constant. In the same way, medium can be transferred between drops, while spheroids remain trapped within the same droplet. Interconnected hanging droplet networks up to 96 wells138 allow intertissue contact and various options for periodic medium exchange and controlled substance exposure, while metabolites remain confined in very small drop volumes around 10 μL.17 This allowed the direct integration of enzyme-based amperometric glucose and lactate biosensors into the droplets by combination with a glass-based sensor chip (Fig. 7B).19 In short-term experiments, within a 20 min static phase, lactate accumulation and glucose production could be measured in parallel (Fig. 6C). Size-dependent metabolic rates from 300–500 μm spheroids in the range of nmol h−1 could be measured. Impedance measurements are also possible. Small tissue size and its position away from the electrodes, which can only be at the ceiling of the droplet, make them challenging however.140 Reducing the droplet volume to bring the spheroid closer to the electrodes for measurement was necessary. Overall, the drawbacks of the hanging droplet approach are the complex liquid handling, necessary prevention of evaporation and limited robustness against mechanical shocks of the systems. Also, the open nature of the principle makes the measurement of dissolved gases, e.g. oxygen, difficult.
Although a number of systems exist with promising new approaches, to date, in 3D cultures, the exact metabolic state and immediate microenvironment, i.e. nutrient and oxygen supply, of the microtissues have still been largely unknown and relied mainly on the controlled exchange of media and diffusion of metabolites. Especially for oxygen, measurement within the microtissue would be desirable, but current microsensors are typically not small enough to be inserted with sufficiently low tissue interference.
The primary advantages of microfluidic systems are the reduction of media volume and thus faster concentration changes, the transport of undiluted medium to downstream sensors, as well as the possibility to repeatedly and efficiently generate a broad range of stimuli. The interplay between the state of the cells, microenvironment and sensing method is complex. Both the choice of a suitable measurement system and the obtained experimental findings must be critically evaluated for the aspects how cells are cultivated and supplied, how metabolites are transported to the sensors and how stimuli are generated. From the perspective of metabolic sensing, it is evident that even in simple, standard cell culture experiments, in which sometimes complex markers or pathways are investigated, basic metabolic parameters at the pericellular level are often unknown. Diffusion of metabolites in stagnant media or even solid matrices is comparably slow. If there is no flow, transport of metabolites to and from the cells will be governed by diffusion and thermal convection only. Therefore, nutrient or oxygen depletion can occur near the cells, even if those parameters seem globally well controlled. In 3D, the state and measurement of metabolites can be even more challenging. As a consequence, this may negatively affect the reproducibility or validity of findings in such experiments. At the moment, microsensors can therefore most likely still contribute more to standardization of cell culture experiments or help in basic physiological investigations, rather than replace classical high-throughput screening approaches.
Bringing sensors close to cells is important in order to obtain pericellular readings. In contrast, the physical presence of a sensor hinders a directly neighboring cell at the position of the sensor and slightly changes the diffusion profile next to the cell. In the case of single cell monitoring, this effect is crucial, and a trade-off has to be made. In 2D or 3D cell cultures, the presence of the microsensor typically does not have much influence, compared to other boundary conditions of the culture itself.
In general, sensors without analyte consumption are preferred to minimize influence on the cellular microenvironment. An exception is the case of 2D cell culture with oxygen sensors positioned between the cells. Typically, cells cannot grow at the very same position of the sensor, thus causing spots in the cell layer without cellular respiration. Therefore, it is beneficial that such a microsensor shows analyte consumption in the range of the cellular respiration to avoid an anomaly in the diffusion profile next to the sensor.
The electrochemical measurement of short-lived reactive species concentration (ROS/RNS) and its spatial distribution is only possible in close vicinity to the releasing cell under stagnant conditions, which excludes downstream detection in microfluidic devices. Planar microdisc sensors in direct vicinity to adherent 2D cell cultures and needle-shaped electrodes, especially suited for 3D culture models, are the only way to address the fleeting existence of these substances. Needle-shaped sensors suffer from bad reproducibility, vibration sensitivity, fragile handling and low throughput due to (motorized) manual positioning under the microscope. Measurement results from planar microsensor platforms with randomly growing cells should be evaluated critically with respect to a particular system design/setup, whose influence on the findings might be underestimated.
Electrochemical, enzyme-based biosensors require frequent calibration because of sensitivity drift, and the enzymatic reaction may release harmful by-products. Placement outside the actual cell culture area addresses both aspects. For long-term application, schemes for recalibration should be considered.
The sometimes considerable complexity of fabrication technology and operation, as well as incompatibility with standard procedures, limits the widespread application of more advanced microfluidic systems. A simplification of microfluidic handling would often be desirable, which could mean the elimination of pumps and reliance on pipetting or passive flow for media exchange. Instrumentation for electrochemical sensors and optical sensors appears to be an expensive investment at first glance. However, upon closer look, the costs for instrumentation equipment are low compared to the typical installation costs for a cell laboratory. Still, there is a need for cost-effective parallelization in order to meet high-throughput screening demands.
Once standardization is established, it is unclear whether the measurement of basic metabolic parameters (O2, pH, glucose, lactate) will be equally important as other indicating parameters like gene expression etc. For 3D cell culture, in which major gradients occur within the microtissues, it is desirable to measure inside such structures with minimal invasion. Instead of sensors, embedded particles which could be read out from the outside are promising alternatives for some parameters and can also address the overall heterogeneity of such cultures.
The basic requirements and challenges for microsensors are the same, independent of whether it is a 2D cell culture or an organ-on-chip system. Notably, the complexity increases from 2D to 3D. Once a system can be used in 3D cell culture models, it can be readily applied to organ-on-chip systems. From the point of view of cell culture monitoring, there is no difference between a 3D culture of cells without organotypic function and an organ-on-chip system. However, the impact of these systems changes from just reporting the metabolic state of the cells to providing insight into cell–cell interactions enabling the functionality of the organ by observing the metabolism of some representative cells within the model. Here, sensor readings can be linked to and provide input for computer models ("in silico"), which become more and more important with the increasing complexity from organ-on-chip to human-on-chip. In our understanding, a meaningful human-on-chip cellular model can hardly be described and accessed without microsensors reading the metabolic state at characteristic points in the system.
|This journal is © The Royal Society of Chemistry 2018


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Most Common Types of Training
Working with your ITI service rep and our engineering team to get your company set up correctly will save you a lot of time down the road. But even if things change — like your company acquires another — Sentix can adapt.
Three Ways to Keep User Lists Updated
With the high turnover at many companies, keeping active user lists can be a full-time job. That's why we strongly recommend connecting Sentix to your back-office systems to avoid double-entry or any other human-introduced errors. Plus it's just easier!
There are three ways you can keep user lists updated:
Manually: Sentix lets you edit any field except the username in the Sentix interface.
Spreadsheet upload: You can upload a spreadsheet into the interface, and it will activate any new users, and deactivate any users it does not find. Accidentally deactivate the wrong person? Don't worry — just upload a new list with them on it.
Users and Roles
Each user in Sentix has a unique role and access level. User restrictions and allowances can be modified as needed — for example, the ability to restrict an admin to just one location.
- Can add Users to the system, including Admins and Read-only Admins
- Have full access to Reporting and Assignments.
- Can access all courses as a user.
- Can print completion certificates.
Read-only Admins:
- Can report and export on activity but can't make changes to assignments or schedules. This stops assignments from being removed, and prevents unauthorized assignments.
- Can access all courses as a user.
- Can print completion certificates.
- Can access all courses as a user.
- Can print their own completion certificates
Using Your Org Chart
Since many assignments and reports are driven by where someone works, or their job title, these form the backbone of the set-up. Each user in the system follows your company's hierarchy.Does your company call them "Areas" instead of "Regions"? We can customize the names for you. Do you have additional levels? Further customization is fast and easy. For each user, you can define one:
Job Title
Update Automatically with Your HR Systems
Sentix can also tie in with your corporate HR systems to update this information in real-time, so you're always up to date with the latest changes.
Tag Them With a Group
Each user can also belong to multiple groups. These are useful for making groups or specializations that don't necessarily fit the other hierarchy.
Example Groups:
- Teams: Maintenance or Dispatch or Account Services
- Certification: Driver Trainer or Senior Dispatcher
- Endorsement: Hazmat
- Life-Cycle: Orientation, Manager Pre-Requisites
- Corrective: HOS Violation, Speeding

subset: commoncrawl


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Photo Credit: http://dianablography.wordpress.com/
So I'm going to tell you something. Don't go getting a big ego about it.
You're beautiful.
Yes, fine, even the guys out there.
I'm not trying to get in your panties/boxers/tighty-whities or anything. I just wanted to tell you because the world doesn't say it enough. The world is saying things like…
Cover up those flaws on your face! Now, because the man of your dreams doesn't like blemishes.
Isn't fast food awesome? Just don't get fat, because you're totally getting fat.
How about a gym membership because you're so fat?
Want to buy some big dick pills? They're awesome at keeping your dick the exact same size.
You're old. You should really get a boob job to make yourself hotter. Sofia Vergara style.
OMG grey (yes, I spell grey with an 'e' instead of an 'a') hair? You better dye it!
Now you're too skinny. Get some butt injections! Twerk, twerk, twerk!
Most of the people saying you aren't good enough are likely trying to sell you something. Either that or they believe the hell out of what's being sold.
Screw. That.
Be you. Yes, there are things that I want to change about myself too. For example, I want to lose weight. But that's more because I want to be healthier as well as challenge myself physically in the near future.
Also, sex is more awesome when your cardio is on point.
I grew up as a kid who totally thought he wasn't beautiful enough. To a certain extent, the world picks up on what you believe about yourself. And then it reinforces that belief. As soon as I started embracing who I am, the world reciprocated and I found others who thought I was beautiful as well.
Yes, beautiful. I don't care if that's not a manly word.
Sure, someone can find you aesthetically appealing, but that's so short-sighted, isn't it?
I think real beauty comes from the energy you exude. Some call it confidence; I call it acceptance. Everyone saying you're not good enough is denying the person you are. Why deny yourself as well? It's funny how explaining this in slightly different words shows how shitty we are to each other sometimes.
Sometimes it feels like a lot of times.
But I suppose that's the price when there's money to be made. I mean, many people are looking for acceptance so it only makes sense someone capitalizes on that, right?
If you say so. In the meantime, I'll be here not caring.
Peace out, party people.


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White April
from: Kayla D.
message: Cathy is amazing with her clients and treats everyone individually. She went above and beyond for me as I had lots of extras I wanted added and she made sure it happened. I'm still blown away at how stunning and how different my dress was :)


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Wednesday, October 1, 2008
The Curb
I called back to Seth, who was behind me on his bike, "Can you do that?"
He pulled up on his handlebars just like I did, but he was too early. His bike hit the curb wrong and he crashed on his side, hitting his face on the concrete.
"No," he said.
Later he accused me of laughing too hard at him, and I reminded him that he'd been laughing, too.
"That's because there was a guy there watching, so I couldn't just lie there and cry," he said.
So I laughed harder. Poor Seth.
5 comments:
- Julie said...
One time, my mom fell down the last couple of stairs, grabbed the banister, and flew headfirst into the wall.
It was the funniest thing I've ever seen.
I don't think she's forgiven my sister and me for laughing so hard.
It was eight-ish years ago. hahaha
- October 1, 2008 at 2:29 PM
- Chrissie said...
Ouchie! You know, I had the exact same experience in Danville. My family had just moved there, and I went biking with my dad. I was about to go on the curb (not even the full-blown curb...it's the part that lowers down to the asphalt so cars can pull into the driveway), but somehow my tire skidded against it in a parallel fashion (not perpendicular like I had hoped). I fell off and scraped my lip and shoulder. Then I had to go wash up in the bathroom of some car shop. After that, I did not have good feelings toward Danville, but I quickly discovered some redeeming qualities, like this girl named Dani who took me under her wing.
- October 1, 2008 at 8:32 PM
- Dave said...
male ego response: Heck yeah Seth had to laugh, but YOU... That's harsh...
- October 5, 2008 at 2:21 AM
- LJ said...
I'm reading this while sitting here in a meeting and had to pinch my nose shut to keep from laughing.
- October 6, 2008 at 12:58 PM
- Seth said...
Dani failed to mention that I said "No" while I was still in mid air.
- October 14, 2008 at 4:04 PM


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What are the Best Debt Consolidation Companies in Foley AL
Who has the best Foley debt consolidation services?
Foley consolidation services offer a way of taking multiple debts and taking them all and wrapping them together into one payment. So if you have 5 different Foley loans, each with a different interest rate and length, a Foley debt consolidation loan will pay these off and then you will be left with one Foley loan with a total on the loan of all the paid off loans. The best part is that these types of Foley consolidation loans typically have lower interest rates than the ones you are getting rid of.
What is the best place in Alabama to find Foley credit consolidation lenders?
Search online for the best debt consolidation lenders in Foley AL. The reason the best place to find Foley debt consolidation lenders in Alabama is because you will be able to find the right Foley lender for you. Your local Foley banks may not specialize in the right Foley consolidation loans for you or have Alabama lending standards that are too stringent. By searching for the best Alabama debt consolidation loans online, you can assure yourself of getting the right Foley loan.
More On Debt Consolidation Loans in Foley AL
As I said above, prior to the credit crunch we had basically "easy money" whereby the Foley banks were encouraged to lend to pretty much anyone in Alabama with a pulse. As the economy tanked and their Foley debts mounted, a large portion of these people who got easy credit are now looking for Foley credit card consolidation programs. These Foley loans can be obtained but they are just like any other Foley loan. Your Alabama credit rating, your ability to repay the Foley loan, an availability of collateral to secure the loan and the current Foley bank policies on Alabama lending will determine if you get a Foley consolidation loan or not.
Where Is The Best Foley Loan Consolidation Service?
As I discussed above, the typical Foley loan consolidation service usually has a lower interest rate than the standard Alabama loan that is putting you in the poor house. Be aware that just because you are in trouble with your Foley monthly payments, does not mean that you will automatically get Foley loan consolidation.
Where Can I find The Best Foley Debt Consolidation Help?
I spoke of collateral before and this is a big aspect of why Foley debt consolidation lenders may give you Foley loan consolidation. Unsecured Foley loans are the riskiest loans to give by a Alabama lender. This is because if the Foley loan is defaulted on, the bank cannot get the money back. By requiring collateral to provide security on a loan (as some Foley banks do for Foley debt consolidation loans) the bank reduces their risk for the loan. A secured Foley loan allows the lender to offer the loan to you and then to offer the Foley loan at a reduced interest rate. Be careful to make sure you pay off the Foley credit card consolidation loan on time. If you were required to put up collateral and you default, the Foley bank will take that collateral as a way to recoup their loss.
Where Can I find The Best Alabama Debt Consolidation Help?
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The deadline to pre-register for the Animal Rights 2019 National Conference is quickly approaching! After July 12, the cost of registration will increase to the on-site registration price of $250. With only 15 days to go…Read more
As we count down the days till #AR2019, We're excited to share the #AR2019 tentative program schedule with you and invite you to join the action July 25-28 at the Hilton Alexandria Mark Center just…Read more
We are exited to announce our preliminary speaker list and share it with you all! We do call it the preliminary because there are still speakers to be added while we work on the program…Read more
We are excited to announce that our speaker applications are now open. You are invited to submit a speaking proposal for the Animal Rights 2019 National Conference which will take place July 25-28 at the Hilton…Read more
We are excited to launched our exhibitor registration, with a new tiered pricing scheme. Pricing is now based on the organization's annual income, with a 15% discount for all nonprofits. Why exhibit at AR2019? It…Read more
You are among the first invited to register for the Animal Rights 2019 National Conference that will take place July 25-28 at the Hilton Alexandria Mark Center, just outside Washington, DC. The first 125 people…Read more
We will be accepting speaker applications beginning Friday, February 15. Our application process will be more in-depth this year, as we are seeking to gather more initial information to make the selection process more efficient.…Read more
If you're wondering what the conference is all about, check out last year's report below: AR2018 Strengthened the Animal Rights Movement "What an amazing, empowering, inspirational event. I love the passion of the individuals and…Read more
Questions? Please contact us here.
Stay up-to-date with new announcements by joining our email alerts list.


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Tag Archives: natural cures for high blood pressure
Home » Posts tagged "natural cures for high blood pressure"
Lower Cholesterol and High Blood Pressure naturally
Many people have asked me how to cure high blood pressure, naturally. Here is the amazing diet that consists of natural cures for high blood pressure. If your Doctor requires you to be on medication, for reasons of safety, you can still try this diet for 6 weeks. Then return to your Doctor, to have […]Read More


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Are you a student in Luxembourg? Seeking a career path?
Meet us, on 26th March 2019.
The event aim to setup a "STUDENT CAREER NETWORK".
Our 1st anniversary
February 8th, 2019, from 7 PM
Come and join us in celebrating our first anniversary!
Vacanz 2019
January 18|19|20, 2019
The tourism fair – Active holidays, wellness and relaxation
"Dear visitors and members,
It is a great pleasure to welcome you at the Chamber of Commerce Albania – Luxembourg, where our aim is to offer outstanding services in assisting your business between our two nations.
We are confident that we will be an invaluable partner in your success."
Artan Vigani
President of CCAL


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Introduction: Akshardham mandir is the biggest temple in India till date. According to previous years records, it attracts more than 60 percent of all tourists who visit Delhi. 141 foot high, 315 foot wide and 371 foot long this beautiful temple Akshardham mandir is located near the banks of the Yamuna in eastern New Delhi. The temple was built according to the Vastu Shastra and Panchtantra Shastra. Complex houses many of things namely Abhisheka Mandap, Sahaj Anand water show, a thematic garden and three exhibitions namely Sahajanand Darshan, Neelkanth Darshan, and cultural boat ride which is also known as Sanskruti Darshan. Temple has idols of the saints of that sect along with other Sanatan dharma gods and goddess. In total, mandir has 234 ornately carved pillars, nine domes, and more than 20,000 murtis of sadhus, devotees such as Sita Ram, Radha Krishna, Shiv Parvati, and Lakshmi Narayan, and acharyas. It also contains 148 life-sized elephants which in total weigh approximately 3000 tons. There is also a theatre there in the complex with having Delhi's largest screen known as Neelkanth Kalyan Yathra. One can see after coming out of the temple a 27 feet tall bronze statue of Neelkanth. Designed in the ancient style of Indian architecture this is not only a temple but a short display of Delhi's culture and spirituality.
History: Yogiji Maharaj was planning the complex since 1968, the spiritual head of the BAPS. But unfortunately, he died in 1971. In 1982 spiritual head of BAPS, Pramukh Swami Maharaj, the successor of Yogiji Maharaj, started working towards fulfilling the dream of his guru. Construction work began on 8th November 2000. The construction work was done in 4 years and 362 days with the help of Maharaj's more than 3,000 volunteers and 7,000 craftsmen. Temple was inaugurated by president of India Dr. A.P.J Abdul Kalam(at that time) on 6 November 2005, prime minister of India Mr. Manmohan Singh and leader of opposition Mr. L.K Advani in front of more than 20,000 guests.
Open: Tuesday to Sunday
- Entry:9:30am-6:30pm
- Exhibition: 10:00AM- 6:00PM
Aarti timings: 9:30am and 6:30pm
Address: Noida mor, Delhi-110092
Nearest metro station: Akshardham


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DGS Publications
|Publication Date
|OFR50 Database of Quaternary Coastal Geochronologic Information for the Atlantic and Pacific Coasts of North America (additional information for sites in Peru and Chile)
|Wehmiller, J.F., Pellerito, V.
|GM16 Geologic Map of the Fairmount and Rehoboth Beach Quadrangles, Delaware
|Ramsey, K.W.
|OFR31 The Storm of July 5, 1989: Hydrologic Conditions
|Talley, J.H.
|RI46 Shallow Subsurface Temperatures at Selected Locations in Delaware
|Woodruff, K.D.
|B16 Ground-Water Resources of the Piney Point and Cheswold Aquifers in Central Delaware as Determined by a Flow Model
|Leahy, P.P.
|OFR18 A Numerical Indicator of Water Conditions for Northern Delaware
|Jordan, R.R., Woodruff, K.D.
|RI9 Ground-Water Levels in Delaware January, 1962 - June, 1966
|Woodruff, K.D.
|B11 Ground-Water Resources of Southern New Castle County Delaware
|Rima, D.R., Coskery, O.J., Anderson, P.W.
|B10 Salinity of the Delaware Estuary
|Cohen, B., McCarthy, L.T. Jr.


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The Dust Palace Productions
By Eve Gordon
I want to embed this investment somewhere
Investment: Art
The Dust Palace Productions: We're a circus-theatre company intent on creating a circus creation space in Auckland, helping Aotearoa's voice to change the world.
by Eve Gordon
Minimum target: NZ $75,000
Days left: Closed
Funded: NZ $140,000
Investment: Art
The Dust Palace Productions: We're a circus-theatre company intent on creating a circus creation space in Auckland, helping Aotearoa's voice to change the world.
by Eve Gordon
Minimum target: NZ $75,000
Days left: Closed
Funded: NZ $140,000
NZ $140,000 pledged
52 people pledged
NZ $75,000 minimum target
Pledges will only be confirmed if the target is reached by: 03/10/2018 at 6:00 PM (NZDT)Make a Pledge
The Dust Palace Productions
The Dust Palace came together almost ten years ago to tell stories using circus arts.
The circus sector has grown and we've grown. We need to expand to meet demand and so we're moving premises to a bigger, better space. We're equity crowdfunding to safely fit-out the new, shared space with the circus gear we need to teach the next generations, expand the circus industry and fulfill our creative potential.
Our mission
To create experiences which change peoples lives by:
- presenting fun exhilarating circus theatre that is intimate, visually stunning and brave in its frankness about human nature.
- setting up a sustainable circus theatre creation space in Auckland, allowing Aotearoa's culture to change the world.
Our story thus far…
The Dust Palace began as a circus theatre company in 2009 with a sell out circus-burlesque show. Knowing that we wanted to tell stories using circus as the medium, founders Mike and Eve thought about the following 10 years closely in terms of how to grow their impact and audience.
Over the following nine years The Dust Palace has produced 13 original full-length circus-theatre works, as well as innumerable other projects creating a large body of work to their name.
To help build our audience, generate revenue and keep their highly skilled performers in the country a corporate performance arm of the business was developed. Immediately claiming the top position in the marketplace, the unique proposition of Dust Palace performance for events has always been quality, aesthetic, and production value.
To build the community and the circus sector as a whole (as well as having space to rehearse) they set up charitable trust along side a school which opened in 2013 as the first circus school and full-time circus facility in Auckland.
The school has founded both a scholarship program, providing underprivileged kids a years' free tuition, and a youth company, giving talented youth tools for creation and performance that will set them apart in future. Being part of the Dust Palace scholarship program or youth company helps young people gain confidence, esteem and give them a place to belong.
Who are we
We're two people, Mike Edward and Eve Gordon. We've worked relentlessly for the last ten years and seen a community build up around us.
Once the company had begun, we grew keenly aware of what it provided to those around us in terms of safety, employment, purpose, joy, creation, and learning… and of our responsibility, our duty of care to keep it existing for those people.
We weren't prepared for the depth of change the Dust Palace would allow people to achieve. We are consistently humbled by the impact the Dust Palace and our community has on so many people. Creating a space in which creativity is honored and valued and inspiration is an everyday state for people to operate from is such a wonderful feeling.
Why we do what we do
We started Dust Palace to start telling stories using circus as a language. We wanted to make a safe space for the misfits of the world to thrive, and fall, and catch each other. We wanted to make shows that made people double-take, at what they saw and at the world around them. We wanted to stretch the corners of that world - of what was deemed possible - and to offer a space for kids to dream.
The dream is to have a thriving hub for circus arts in Auckland in order to grow the art form in innovation, attention and engagement.
We have found another building in our area which is entirely ideal; at three times the size of our current space, and with three meters more height, it has a superior structure for the rigors of circus. On top of this, it has extra space for onsite physiotherapy/acupuncture/massage facilities.
The opportunities of the new space vastly outnumber those of our current facility; we will finally be able to offer team building workshops for corporate clients and birthday parties for kids. We will set it up for a range of performance projects and it is large enough to offer as an event venue.
In addition to growing the range of activities, the level of skills we will be able to offer through tuition will increase (new apparatus and higher level technique). We will be able to attract international trainers if our facility is world class.
The plan is to lessen the risk by sharing the space with other like-minded companies; other circus companies, dance companies, and stunting companies. Through sharing resources we will create a vibrant hub of a training space.
Why we're crowdfunding
The Dust Palace has chosen to equity crowdfund our next stage because crowdfunding is inclusive.
In a nutshell, we've outgrown our space; our classes are full, the professional team has to book time slots to rehearse, and we are producing new shows that we simply cannot develop in our current facility.
However, we find ourselves in the catch 22: To relocate and fit out a bigger space we need capital, to generate capital we need a bigger space.
A larger venue helps us become sustainable. With twice the classes, a range of new income-generating activities, and a new space-share model, we will be able to employ support staff and administrators. We will have the opportunity to create a legacy space for circus in Auckland.
Selling shares in the Dust Palace is about building community ownership. We have come a long way on our own, but with further support, we know the possibility of building a large and sustainable circus facility together is very real.
We have found an opportunity to relocate for 2019 but we need your help and support to do so.
We have become aware that the ownership of a company created for its community should be non-hierarchal and, therefore, open to the people of this community. As such, we would like to engage with both our audience and students alike to allow them to tangibly partake in what is ostensibly already theirs, and, the future of the very thing they love.
Opening a new, bigger, hub-style space will allow the industry to believe in an inclusive future where circus resources are shared and a melting pot of amazing creative energy is possible.
The benefit of getting more people involved in the infrastructure of the circus community will inspire further advocacy and championing of the sector as a whole. Sustainability, continued growth and constant, unique creation will be a few key features of a greater shared ownership. More numbers on the ship will allow the Dust Palace as a whole to steer in the right, safest and most supportive direction for all.
This project will strengthen our place in the competitive marketplace, launch an exciting new venue, and offer our shareholders the opportunity to join a growing business in a growing community.
In order to fit out the new space and survive the initial growth period we need a minimum of $75k.
The Share Offer
Rights attached to the shares
Investments under $10,000 will purchase non-voting shares in The Dust Palace Productions Limited. Those over $10,000 will be granted Investor shares with full voting rights.
The rights attached to both types of shares are outlined in the Rights attached to the shares section in our IM.
Use of Capital
About our Team
Structuring a venture like this correctly is vital. We have been planning, adjusting and dummy-running systems all year in order to have them functioning perfectly should we open in January 2019. Health and Safety systems have been audited, we've been testing and adjusting our online booking system to make sure it's going to function perfectly for a complex and busy space, we have addressed the overall structure of the Dust Palace eco-system, created new roles, shifted others and made sure all areas are being accounted for, especially in financial support and revenue development.
What we've done so far
We've grown from a group of friends putting on fringe shows to being asked to perform our works internationally, being asked of collaborate with the Auckland Philharmonic and filling the Manawatu Arena with an original Dust Palace work.
"Breathtakingly beautiful. Heart-stoppingly romantic. Stirringly erotic" — Jo Ledingham, Vancouver
"Immediately awesome! A full house, standing ovation and the most common word on everyone's lips: Spectacular!" — Janet Whittington, Theatreview,
"When two powerhouses such as these two come together, you just know you're in for an incredible theatre experience…. …This enthralling collaboration is a beautiful celebration of circus and symphony deserving of a full season as put simply it is an experience that needs to be seen far and wide." Faith-Ashleigh Wong, Appetite for the Arts
Here is a little run down of the last ten years in brief:
What's next?
Open a Circus Creation Space with a functioning Shared Model
Provided the equity is raised 2019 will see us open the first ever shared model circus creation space in Aotearoa. Companies will be able to contribute and use the space in ways that work for small arts groups and independent companies. Due to the increased facilities we'll be able to produce even more amazing shows and give our students the opportunity to learn a range of different skills, beginning NZ on our journey to being internationally competitive.
Develop Circus as an Art Form in Aotearoa.
The new space will allow us to both expand the reach of our school and also offer outreach programs and engage in audience expansion. The more people engaged with circus at a young age means the audiences of the future will be greater and more invested in the art form.
Build a Legacy Company with an International Quality Touring Repertoire.
Ensuring the structure of the organisation is sustainable and best practice is always upheld in business, ethics and health and safety will enable The Dust Palace to achieve a greater height of artistic merit. Creating national and international touring circuits will cement the value and place of our shows in the consciousness of Aotearoa.
Risks and Challenges
With any endeavor, it is vital to explore the risks and commit to practices that will mitigate those risks. Below are the key risks we have defined and our plans around mitigation outlined in brief.
Not achieving the student numbers or space hire to service the rental.
- Sharing the space.
- Marketing strategy.
- Business advisory board.
- Re-structure and role definitions creating a wider base of support for the organisation.
Fatality or major incident in the industry
- Leading H&S systems for circus in Aotearoa.
- Prioritizing a dedicated and permanent H and S role within the core team.
- Supporting ANZCA and advocating for best practice.
- Being consistently vocal about safety first mentality.
Loss of key person; Mike or Eve.
- Prioritising fiscal remuneration for key roles.
- Allowing Mike and Eve enough time holiday and connect.
- Spreading the base of operations and knowledge.
- Formalising operations, values and procedures into written documents.
Growing too fast for current resources.
- Making sure their are clear and valued pathways for teachers and performers.
- Recognising and cultivating talent early on.
- Strict forward planning.
Note from PledgeMe
We have carried out a credit check on the company and it's directors, as well as a Google check. Here are our findings:
- Director, Mike Edward, filed for voluntary bankruptcy in 2012 and was discharged in 2015. More info can be found on the Insolvency Register (estate no. 854376)
We got there!
19/09/2018 at 11:29 AM
Hi everybody!
It is with great pleasure that I write to announce that we have achieved our maximum investment target.
52 wonderful people are now well and truly a part of the next phase of the Dust Palace. Thank you from the bottom of our hearts.
Achieving the maximum means that the fit-out we once only dreamed of is now entirely a reality: we will have a fully sprung dance floor, all the best new equipment and gear, and the most lush foyer/reception area you could hope for. The new space will be nothing short of first class. It will be comparable , if not superior, to competitive facilities we have seen offshore.
Next steps from PledgeMeNow that the campaign has closed, we'll be quiet for the next three weeks, as Team PledgeMe work hard to verify your details and collect your pledges, before transferring the funds to us so that we can put it to work!Here's the steps in their process:
- for those who are investing through PledgeMe for the first time, you'll have received an auto-email letting you know that PledgeMe are carrying out your investor identity and address check. For those who have have been verified previously, you will have received the payment details to complete your investment
- if they need to clarify either your identity or address information, Team PledgeMe will be in touch directly with you in the next four working days
- Once verified, you'll receive the payment instructions to complete your investment
- If you haven't heard anything from PledgeMe by Wednesday 26th September, please check your junk for messages from [email protected], and if there's nothing there, please let [email protected] know.
Thank you all so much. We are truly humbled. We are overwhelmed. But I can also assure you that we do not see this as the end... we see this as the beginning.
Eve and I have a drive to make every cent of everyones investment worth so much more. We are ambitious and will work relentlessly to keep pushing our work and upholding the highest possible standards within the industry.
We intend to become truly international. We want to lead the circus movement in Aotearoa and we seek to create something that will last well beyond all our time.
So thank you from the bottom of our heart for your support. Now sit back, and watch us fly.
Best Regards,
Mikey and Eve
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Offer Details
|Current Valuation
|Raise Minimum
|Raise Maximum
|Share Price
|Maximum Shares Offered
Explanation of valuation:
Financial Summary
|Prev Year
|Current Year
|Est. FY 2021
|Est. FY 2022
|Operating Expenses
|Net Profit
Company Details
Company Name: The Dust Palace Productions Limited
Company Number: 9429031691581
Company Documents
Director Details
|Profile URL
|Eve Gordon
|Mike Edward
Ask a Question (You must login to ask a question)
Realistically, what are the chance of dividends in the next 10-15 years? Wouldn't you invest back into the company first? Or would you like to reward investors beyond the pledge-me benefits?Posted on 06-09-2018 by Steve King
Hi Steve! The plan is to re-invest profits during the growth phase (3-5yrs). But after the 5 yr mark we are confident the business will then be running at a profit and dividends will be able to be distributed amongst shareholders.
Answered on 06-09-2018 by Eve Gordon
Is the "free open training" transferable? As a 52yo male I'm unlikely to take up the offer but may wish to offer it to a younger relative or other worthy young person.Posted on 06-09-2018 by Steve King
Yes. All our rewards are transferable once only on purchase. So as much as we think you would probably love to do some aerial work we appreciate that you may have another who might enjoy it more...
Please note that the rewards are not transferable at any stage after the initial purchase: If shares are sold in the future the reward remains with the initial holder.
Answered on 06-09-2018 by Eve Gordon
Can I change my pledged amount?Posted on 18-09-2018 by Rosalind Giffney
Hey Rosalind! I'm finding out for you now. If you want to msg or email me the details that would be amazing too!
Answered on 18-09-2018 by Eve Gordon
Hi, We enquired with pledge me about increasing our pledge amount but haven't got an answer regarding this yet, please can you let myself or Bernard know? ThanksPosted on 18-09-2018 by Annuskha Dunstan
Followers of The Dust Palace Productions
2018-09-06 09:07:46 +1200
2018-09-04 22:24:15 +1200
"I love circus and the DP teachers are the best!!! Here is a chunk of my nest egg which I promised my partner I wouldn't spend 😝😮 "
2018-09-04 19:52:41 +1200
"Eve & Mike - We're looking forward to seeing the next leaps in your journey"
2018-09-04 15:57:29 +1200
"No comment 😂"
2018-09-04 12:13:45 +1200
"I love the Dust Palace and what it stands for. I have even had the pleasure of working with their company on more than one occasion, and would love to be able to maintain such a professional relationship on as many levels as possible. It is clearly a leader in its field, Circus Theatre."
2018-09-04 07:35:38 +1200
2018-09-03 23:12:07 +1200
2018-09-03 22:25:09 +1200
"Good luck, hope the campaign goes well, and looking forward to seeing what the future brings for Dust Palace."
2018-09-03 21:15:42 +1200
"Good luck with this raise and your drive to take the Dust Palace where it belongs."
2018-09-03 21:03:07 +1200
"I have been drawn to the beautiful people in the Dust Palace since I met you all on your launch night many years ago and with every venture seen you grow and amaze. I look forward to seeing you continue to shine brighter and bring beauty to the world. It is an honour to be included......xo "
2018-09-03 20:38:22 +1200
2018-09-03 17:38:57 +1200
"Thank you from the bottom of my heart for creating a space where I have learnt so much, built strength, and filled my soul with joy. I have changed and grown for the better on a personal level too. What a wonderful community you have created! I am so grateful and can't wait to continue the awesomeness in the new space!! "
2018-09-03 11:23:50 +1200
"It is a pleasure to invest in and to support such an amazing group."
2018-09-03 09:53:54 +1200
2018-09-03 09:29:38 +1200
"Rule no. 13"
2018-09-03 09:28:39 +1200
2018-09-03 09:28:30 +1200
2018-09-03 09:18:25 +1200
2018-09-03 09:16:47 +1200
"(: "
2018-09-03 09:11:10 +1200
2018-09-03 09:10:57 +1200
2018-09-03 09:10:10 +1200
"Go go team DP! "
This campaign has closed, but this company may choose to do more equity raises on PledgeMe in the future. If you're interested in investing in The Dust Palace Productions, you can sign up to be notified when a new equity campaign from this company is published.
Investor Perks
Investment $505 – $999
Investor shares | Foundation brick on the wall | 10% discount on classes at the Dust Palace for life or 10% discount on show tickets for life (when available)
Investment $1000 – $1999
Investor shares | Foundation brick on the wall | Invitation to exclusive bi-monthly event | 20% discount on classes at the Dust Palace for life or 20% discount on show tickets to shows for life (when available)
Investment $2000 – $4999
Investor shares | Foundation brick on the wall | Free open training for life | 30% discount on classes at the Dust Palace for life | preview show tickets for life (when available) |VIP membership to exclusive bi-monthly invite-only speak-easy performance event
Investment $5000 – $9,999
Investor shares | Foundation brick on the wall | Lifetime membership (includes classes and open training – can be gifted* terms apply) | Opening night tickets to shows for life (when available) | VIP membership to exclusive bi-monthly invite-only speak-easy performance event
Investment $10,000 +
Full voting shares | Invitation to board of directors meetings | Foundation brick on the wall | Lifetime membership (includes classes and open training – can be gifted* terms apply) | pening night tickets to shows for life (when available) | VIP membership to exclusive bi-monthly invite-only speak-easy performance event | A bespoke performance for your event (*terms apply)
I want to embed this investment somewhere
Investment: Art
The Dust Palace Productions: We're a circus-theatre company intent on creating a circus creation space in Auckland, helping Aotearoa's voice to change the world.
by Eve Gordon
Minimum target: NZ $75,000
Days left: Closed
Funded: NZ $140,000
Investment: Art
The Dust Palace Productions: We're a circus-theatre company intent on creating a circus creation space in Auckland, helping Aotearoa's voice to change the world.
by Eve Gordon
Minimum target: NZ $75,000
Days left: Closed
Funded: NZ $140,000