Human in vitro physiological models studying disease and drug treatment effects are urgently needed as more relevant tools to identify new drug targets and therapies. We have developed a human microfluidic two-organ-chip model to study pancreatic islet–liver cross-talk based on insulin and glucose regulation. We have established a robust co-culture of human pancreatic islet microtissues and liver spheroids maintaining functional responses up to 15 days in an insulin-free medium. Functional coupling, demonstrated by insulin released from the islet microtissues in response to a glucose load applied in glucose tolerance tests on different days, promoted glucose uptake by the liver spheroids. Co-cultures maintained postprandial glucose concentrations in the circulation whereas glucose levels remained elevated in both single cultures. Thus, insulin secreted into the circulation stimulated glucose uptake by the liver spheroids, while the latter, in the absence of insulin, did not consume glucose as efficiently. As the glucose concentration fell, insulin secretion subsided, demonstrating a functional feedback loop between the liver and the insulin-secreting islet microtissues. Finally, inter-laboratory validation verified robustness and reproducibility. Further development of this model using tools inducing impaired glucose regulation should provide a unique in vitro system emulating human type 2 diabetes mellitus.
Human long-term organoid cultures in microfluidic microphysiological systems (MPS; or organs-on-a-chip) can emulate human biology and, therefore, enable detailed temporal studies of the physiological functions of organs, organ cross-talk and pharmacological effects of drugs1,2. One major goal of MPS efforts is to recapitulate a disease state and study the effects of drug treatment. Such models have the potential to transform drug discovery, enabling efficient studies, and potential identification, of new drug targets, as well as testing of drug interventions in physiologically relevant models. Several recent publications have shown considerable progress in developing relevant human models, such as human single-organ lung-on-a-chip3 or liver-on-a-chip platforms4. Further development into disease models is progressing for lung equivalents5 and for liver equivalents6,7,8.
However, systemic human diseases progress through the disruption of the homeostatic cross-talk of two or more organs. To emulate such systemic interactions, a few microphysiological platforms are aiming to develop on-chip co-cultures of different organoids in separate culture compartments, interconnected through microfluidic channels9,10,11,12,13,14. A first organotypic homeostatic long-term co-culture of human skin biopsies with three-dimensional (3D) human liver spheroids has been established successfully on a multi-organ chip (MOC) platform15. Subsequently, MOC-based long-term co-cultures of liver spheroids with human 3D intestinal16 and neuronal17 tissue models were established for systemic repeated dose substance testing.
Type 2 diabetes mellitus (T2DM) is increasing in incidence and associated with multi-organ co-morbidities18,19,20. As insulin resistance develops, the pancreatic islets meet the increasing demand for insulin by increasing secretion and expanding islet mass. It is only when islets fail to adapt that blood glucose levels increases and overt T2DM develops. As insulin is a key regulator of hepatic metabolism, shifting the balance from glucose production to favour glucose storage, hepatic insulin resistance contributes to impaired glucose homeostasis defining T2DM. A wide range of animal models displaying various characteristics of T2DM has been generated in the past21. Despite this, extrapolations from animal research have resulted in a poor translation of studies to understand and improve glucose metabolism, from molecular mechanistic findings through phenotypic manifestation and progression of disease to pharmacological effects of novel therapies22. Importantly, many of the anti-diabetic drugs currently in clinical use, though effectively treating symptoms, have limited effects on disease progression. Microphysiological co-culture systems of human pancreas and liver, which are two of the key organs involved in maintaining glucose homeostasis, might provide a unique platform for emulating T2DM. Consequently, our aim was to establish a human two-organ-chip (2-OC) model replicating long-term physiological cross-talk between pancreatic islet microtissues and liver spheroids.
The pancreatic islets of Langerhans are endocrine organelles scattered throughout the exocrine portion of the pancreas. They are comprised of at least five distinct cell types: α cells (secreting glucagon), β cells (insulin), γ/PP cells (pancreatic polypeptide), δ cells (somatostatin) and ε cells (ghrelin). Isolated pancreatic islets represent a useful model for studying islet function and hormone secretion. As of today, MPS have only been used for short-term cultures of pancreatic islet microtissues to evaluate hormone secretion profiles23,24 and interaction with endothelial cells25. The 3D InSight™ pancreatic islet microtissues (InSphero AG, Switzerland) used in this study are produced from reconstituted dispersed human pancreatic islet cells retaining the composition of α, β and δ cells representative of normal human pancreatic islets. The 3D islet microtissues maintain normal insulin secretion response to a glucose load in long-term culture for up to four weeks26,27,28.
In the liver, insulin stimulates glycogen synthesis and inhibits glycogen breakdown. It also stimulates glycolysis and inhibits gluconeogenesis. In addition to its effects on glucose metabolism, insulin has a variety of anabolic actions in the liver, stimulating lipid synthesis and release, protein synthesis, and inhibiting the breakdown of these substances29,30. Several studies have explored various platforms to build human liver organ models31. Major efforts in the area have been directed towards drug metabolism and toxicity studies using primary human liver cells or hepatoma cell lines. In this study, we used spheroids formed from HepaRG cells, a carcinoma cell line exhibiting many features similar to primary human hepatocytes, and has been used successfully in drug metabolism and toxicity studies32,33. HepaRG cells were also found to be useful in studies inducing steatosis by lipids34 or drugs35, indicating their usefulness as a metabolic disease model. HepaRG cells have been demonstrated to form 3D spheroids in static culture36, with improved functional performance over a culture period of up to 21 days37. Furthermore, including extracellular matrix producing stellate cells is essential for the formation and long-term preservation of HepaRG liver spheroids11.
We co-cultured human liver spheroids composed of HepaRG cells and primary human stellate cells with human pancreatic islet microtissues to model their physiological cross-talk. In particular we wanted to establish physiological regulation of circulating glucose driven by the biological source of insulin – the pancreatic islets. Reciprocally, we intended to investigate the effect of glucose on insulin secretion. Experiments were conducted in two independent laboratories to establish inter-laboratory reproducibility. The latter is the basis for further development into a reliable human T2DM model.
A 2-OC enabling the culture of human pancreatic islet microtissues and liver spheroids was used (Fig. 1A and B). The tissue compartments and the interconnecting microfluidic channel contained a total media volume of 610 µl. A peristaltic on-chip micropump enabled a continuous pulsatile flow with a pumping frequency of 0.475 Hz and a pressure of 500 mbar resulting in an average flow of 4.94 µl/min with a complete media turnover time of approximately 2 h38, supporting high tissue perfusion rates at pulsatile flow. Pancreatic islet microtissues and liver spheroids were adapted to the medium routinely used for the liver spheroids devoid of the customary supplement of insulin. Ten pancreatic islet microtissues were placed in the compartment upstream of a chamber containing 40 liver spheroids (Fig. 1C). This is equivalent to a factor of 100,000 present in a normal human pancreas (estimated to contain on the order of one million pancreatic islets). The number of liver spheroids represents a similar fraction of a normal human liver.
The microphysiological two-organ-chip (2-OC). (A) A 3D view of the assembled device including temperature support (red). (B) Illustration of the view from underneath with media circuits, respective culture compartments and micropump valves highlighted in red. (C) Standard tissue loading scheme of organ equivalents for 2-OC co-culture. (D) 15-day co-culture schematic: repeated total media exchange (double arrows, 300 µl from each compartment), additional sampling at 0, 8, 24 and 48 h for a glucose tolerance test (grey arrows, 15 µl from each compartment).
A robust 15-day co-culture process was developed and used in two independent laboratories. This included tissue loading, seven complete media exchanges and terminal tissue extraction for further analysis to evaluate the long-term reproducibility of the organ cross-talk (Fig. 1D). Glucose utilisation was evaluated as a measure of organ cross-talk by raising the glucose level in the culture medium to 11 mM mimicking the standard glucose tolerance test (GTT) performed in humans. Subsequently, samples were collected at consecutive time points (Fig. 1D) for measurements of glucose and insulin in the circulating media.
Both the functionality and morphology of representative organoid equivalents were evaluated before their transfer to the 2-OCs. The presence of insulin-producing β cells and glucagon-producing α cells in the islet microtissues was confirmed by immunohistochemistry (Fig. 2A). In addition, all preparations of islet microtissues (n = 3 donors) were confirmed to secrete insulin in response to glucose (Fig. 2B) prior to use. In liver spheroids, the even distribution of stellate cells expressing vimentin across the hepatocytes expressing cytokeratin 8–18 was shown (Fig. 2C). Furthermore expression of characteristic hepatocyte markers was confirmed by staining for albumin and CYP3A4 (Fig. 2E). The insulin receptor was found to be expressed in liver spheroids at approximately 70% compared to primary human hepatocytes (Fig. 2D). HepaRG cells were the only source for that expression as no expression was detected in stellate cells. Finally, functionality of the liver spheroids was evaluated by measuring Akt phosphorylation as a marker of insulin-receptor signalling (Fig. 2F). No significant difference in Akt phosphorylation was observed between insulin-stimulated (white bars) and unstimulated (black bars) cells in monolayer cultures of either HepaRG cells or stellate cells. By contrast, the spheroid cultures showed an insulin-dependent Akt phosphorylation. Interestingly, this was due to markedly reduced basal Akt activity compared to monolayer cultures (0.04 ± 0.02 vs. 0.21 ± 0.01 pAkt473/totalAkt).
Status of pancreatic islet microtissues and liver spheroids prior to co-culture. (A) Protein expression of insulin (red) and glucagon (green) in pancreatic islet microtissues. Nuclei stained with DAPI (blue). Scale bar: 50 µm. (B) Glucose-stimulated insulin secretion (GSIS) from single islet microtissues in low (2.8 mM, black dots) and high (16.8 mM, white dots) glucose. (C) Protein expression of cytokeratin 8/18 (green) and vimentin (red) in liver spheroids. Nuclei stained with DAPI (blue). Scale bar: 50 µm. (D) Insulin receptor mRNA expression in primary human hepatocytes, liver spheroids and stellate cells alone, normalized to TATA-box-binding protein, n = 4 for liver spheroids and stellate cells, n = 2 for primary human hepatocytes. Data are means ± SD. (E) Protein expression of albumin (green) and CYP3A4 (red) in liver spheroids. Nuclei stained with DAPI (blue). Scale bar: 50 µm. (F) Phosphorylated Akt (pAkt, serine-473) normalized to total Akt in unstimulated (black bars) and insulin-stimulated (1 nM, white bars) conditions. *p < 0.05 using unpaired two-tailed Student t test. Data shown as mean + SD, n = 3.
Following the initial characterisation of the composition and functionality of both islet microtissues and liver spheroids, co-cultures (islets + liver) and single cultures (islets only and liver only) were set up and maintained in the 2-OC for 15 days in a common medium. The co-cultures (Fig. 3A) showed stable, reproducible circulating insulin levels (4.3 ± 1.1 nM) over the entire 15-day culture period. Insulin levels in the islet single cultures were less stable, declining over time with an overall reduction from day 3 (11.08 ± 0.81 nM) to 15 (5.66 ± 0.74 nM) of 49%. Following the 15-day cultivation period, the islet microtissues were removed from the 2-OC and GSIS was measured (Fig. 3B). Whereas the islets maintained in co-culture with liver spheroids maintained glucose stimulated insulin secretion over time, islet microtissues in single cultures had a significantly decreased stimulated insulin secretion compared to day 0. Finally, the islet morphology was not affected in 2-OC culture and both β and α cells were still present with no apparent difference in relative distribution compared to day 0 (Fig. 2C).
Pancreatic islet microtissues stay functional in co-culture with liver spheroids over 15 days. (A) Insulin accumulated over 48-h periods measured in the media of co-cultures and islets alone. Data shown as mean + SD, n = 5. (B) GSIS of 11 single islet microtissues in low (2.8 mM, black dots) and high (16.8 mM, white dots) glucose. *p < 0.05 using ANOVA with Tukey's multiple comparison post hoc test. (C) Protein expression of insulin (red) and glucagon (green) in islet microtissues at day 15 in co-culture. Nuclei stained with DAPI (blue). Scale bar: 50 µm.
Insulin secretion by the islet microtissues promoted glucose utilisation by the liver spheroids, demonstrating functional cross-talk between the tissues in co-culture. In single culture, the liver spheroids maintained a stable circulating glucose concentration close to 11 mM (the concentration in the culture medium) over the 15 days in the 2-OC (Fig. 4A, black bars). In co-culture with islets (white bars), on the other hand, the circulating glucose level was reduced within 48 h to a concentration equivalent to normal postprandial levels in man (e.g. 5.9 ± 0.6 mM on day 3), indicating that insulin released by the islet microtissues stimulated glucose uptake by the liver spheroids. However, the ability of the liver spheroids to utilise glucose in the co-cultures decreased over time relative to day 3, but remained stable from day 5 to 15 at levels of 8.3 ± 0.87 mM. Albumin in the circulating media was measured to address whether the reduction in glucose utilisation was due to an impaired function of the liver spheroids. Albumin was produced stably (9.5 ± 1.5 ng in 48 h) in co-cultures and did not correlate with the decreased glucose consumption. Moreover, the histology was inconspicuous and showed intact tissue morphology at day 15 (Fig. 4C). Stellate cell and HepaRG ratios were unchanged compared to day 0 (Fig. 4D), as was albumin and CYP3A4 expression (Fig. 4E).
Long-term functionality of liver spheroids. (A) Glucose levels 48 h after medium exchange in co-cultures compared to liver spheroids alone. Dotted line indicates glucose concentration of the medium applied. *p < 0.05, ***p < 0.001, ****p < 0.0001 using ANOVA with Dunnett's multiple comparison post hoc test. Data shown as mean + SD, n = 5. (B) Albumin production over 48 h in co-cultures (white bars) and single cultures (black bars). Data shown as mean + SD, n = 5. (C) Liver spheroids stained with haematoxylin and eosin after 15 days of co-culture. (D) Protein expression of cytokeratin 8/18 (green) and vimentin (red) in liver spheroids after 15 days of co-culture. (E) Protein expression of albumin (green) and CYP3A4 (red) in liver spheroids after 15 days of co-culture. Nuclei stained with DAPI (blue). Scale bar: 50 µm.
The response to a high glucose load was studied in more detail on day 1, 7 and 13 of culture to investigate the impact of insulin secretion on glucose utilisation. An in vitro GTT was initiated by instantaneously raising the glucose levels to 11 mM by a complete medium exchange. Samples were taken immediately (0 h) and after 8, 24 and 48 h, only causing a reduction of culture volume of less than 10% in total. In contrast to single cultures, the glucose level in co-cultures decreased from the hyperglycaemic to a more normoglycaemic range (3.9 to 7.8 mM) within 24 h on the first day of GTT. Subsequently, glucose levels were balanced in the physiological range without dropping to a hypoglycaemic state. Without the insulin-stimulated glucose uptake, glucose levels in single cultures (islets only and liver only) remained within the hyperglycaemic range (Fig. 5A). Differences in glucose utilisation between co-cultures and single cultures were estimated by calculating the area under the glucose response curve (Fig. 5B), which was significantly decreased in the co-cultures.
Functional cross-talk between liver spheroids and pancreatic islet microtissues. Glucose level during in vitro GTT initiated on day 1 (A), 7 (E) and 13 (I). Dotted lines indicate physiological postprandial glucose range (3.9–7.8 mM). Area under the glucose response curve for day 1 (B), 7 (F) and 13 (J). Insulin level during in vitro GTT initiated on day 1 (C), 7 (G) and 13 (K). Area under the insulin response curve for day 1 (D), 7 (H) and 13 (L). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 using ANOVA with Tukey's multiple comparison post hoc test. Data shown as mean + SD collected from n = 14 from three independent experiments with three donors of pancreatic islets run at two different laboratories in the first lane and n = 5 from one pancreatic islet donor in the second and third lane.
Insulin levels in the circulating media during hyperglycaemia rose steadily in the co-cultures and reached a steady state once glucose levels had dropped to normoglycaemic levels (Fig. 5C). Islet microtissue single cultures had a significantly higher insulin secretion (AUC) over 48 h than co-cultures (Fig. 5D). The islet–liver cross-talk observed on day 1 was highly reproducible across experiments with islet microtissues from three different donors performed in two independent laboratories.
Islet–liver cross-talk was maintained over the entire culture period, with similar trends in glucose utilisation and insulin secretion on day 7 and 13. However, compared to day 1, glucose concentrations reached the normoglycaemic range later, between 24 and 48 h after glucose load. Single cultures never reached the normoglycaemic glucose levels at any of the time points tested (Fig. 5A,E and I). The area under the glucose response curves for the co-cultures were significantly reduced compared to single cultures at all time points (Fig. 5F and J). Insulin concentration profiles in the co-cultures were as well comparable to day 1 showing an increase between 0 and 24 h, and a steady state between 24 and 48 h (Fig. 5G and K). The ability of the islet single cultures to maintain insulin secretion in hyperglycaemia decreased with time, leading to no significant difference in the area under the insulin response curve between co-cultures and single cultures on day 13 (Fig. 5L).
T2DM is a multi-organ disease. Disease phenotype and response to treatment effects are dependent on both fully metabolically functional organs and a relevant interplay between them. Here we present a model of organ cross-talk between pancreatic islet microtissues and liver spheroids, two key organs involved in glucose homeostasis. We have used 3D organoids, which are the smallest fully functional building blocks of the corresponding human organs, that are genetically encoded to self-assemble in a proper environment. The islets and the lobules are the functional units of the human pancreas (endocrine portion) and human liver, respectively. The cross-talk between islets and liver is an important mechanism contributing to normal regulation of blood glucose levels. When stimulated by glucose, the pancreatic islets secrete hormones that drain into the hepatic portal system, where glucagon and insulin affects the liver either to take glucose up for fuel storage as glycogen, or to produce and release glucose as a fuel source to support peripheral organs. We here exposed liver spheroids matching ten human liver lobules to ten pancreatic islet microtissues matching one million pancreatic islets in a MPS. Thereby, the respective human counterpart organs – the endocrine part of the pancreas and liver – were downscaled by a factor of 100,000. This allometric scaling will support quantitative in vitro to in vivo extrapolations using physiologically based modelling and kinetic studies of pharmacology.
The present study resulted in a novel and robust microfluidic chip-based co-culture of human pancreatic islet microtissues and liver spheroids with reproducible organ cross-talk maintained over a period of 15 days in a standardized insulin-free cell culture medium. We established a surrogate functional GTT for the pancreatic islet–liver co-culture simulating the standard intravenous GTT; the gold standard method to simultaneously evaluate insulin secretion and glucose elimination in humans39,40,41. Functional coupling of liver and pancreatic islets was evaluated by measuring the cumulative insulin in media released from the islet microtissues in response to a glucose load. Insulin increased glucose uptake by the liver spheroids accelerating the rate of glucose disappearance from the media. Glucose always returned to normal fasting levels in the co-cultures, but not in the single organ cultures (Fig. 3A,E and I). Thus, insulin secreted into the circulation stimulated glucose uptake by the liver spheroids, while the liver alone did not consume glucose as efficiently. As glucose levels fell, the accumulation of insulin in the media decreased and by 48 h, both glucose and insulin had reached steady state levels, supporting a homeostatic feedback loop between islet microtissues and liver spheroids. Exposed to high glucose over time, islet microtissues cultured in the absence of liver spheroids had reduced ability to release insulin, indicating that the prolonged hyperglycaemia impaired islet function. The results, thus, indicate that the glucose regulation in the 2-OC reflects human glucose homeostasis with adequate insulin response with islet functionality only maintained in co-cultures. However, kinetics of glucose disposal in the 2-OC was considerably slower than in vivo, most likely due to the absence of the major glucose-consuming organs and tissues, such as muscle42, brain43 and kidney44, as well as endothelial45, stromal and immune46 cells in the chip. Furthermore, the media turnover time of 2 h in the 2-OCs might have had an impact on that delay.
Furthermore, we hypothesise that already the hyperglycaemic periods of < 24 h could have induced a lipid-associated insulin resistance in the co-cultured liver spheroids, leading to the decline in glucose utilisation observed over time. In particular, Davidson et al.47 have demonstrated an onset of insulin resistance in primary human hepatocytes maintained in 25 mM glucose within six days of culture. Furthermore, increased lipid accumulation was seen already at 12.5 mM glucose.
Immunohistological staining before and after co-culture showed that pancreatic islet microtissues and liver spheroids maintained normal morphology with stable expression of insulin and glucagon in the pancreatic islets (Figs 2A and 3C) and Cytokeratin 8/18, vimentin, albumin and CYP3A4 in the liver spheroids (Figs 2C,E and 4D,E) over the entire co-culture period. Furthermore, these images depict a stable distribution of the different cell types of the heterologous organoid equivalents – α and β cells in pancreatic islet microtissues and HepaRG and stellate cells in the liver spheroids – suggesting viable but non-proliferating cells in the spheroids. Even distribution of vimentin, an extracellular matrix protein expressed by stellate cells, illustrates continuous connective tissue activity in liver spheroids during the 15-day co-culture. In addition, the stable secretion of albumin and retained CYP3A4 expression support a physiological phenotype of the liver spheroids. Activation of AKT by insulin via the insulin receptor in the HepaRG spheroids containing stellate cells prior to chip loading demonstrated the physiologically relevant responsiveness of the liver spheroids (Fig. 2F). The ratio between stimulated and unstimulated AKT phosphorylation was considerably higher in the liver spheroids compared to monolayer HepaRG cells, showing that the 3D culture setting is advantageous for a physiological response.
These results now encourage us to induce T2DM conditions in the co-culture, subsequently leading to characteristics of T2DM in the 2-OC model established. The islet–liver culture presented here can, thus, be used to unravel mechanisms and comorbidities associated with T2DM disease progression including β-cell failure, hepatic insulin resistance, steatosis, steatohepatitis and cirrhosis. An additional development of the MOC model would be to include other organs, such as kidney, cardiac tissue and fat, or different combinations of organs, dependent on the scientific issues being investigated. A future improvement of the model would be to replace the HepaRG cells with primary human hepatocytes to reflect the functions of a human liver better and to select specific pheno- and genotypes.
The microphysiological 2-OC, described earlier by Schimek et al.48, consists of two spatially separated compartments for the cultivation of organ equivalents (Fig. 1B), which are interconnected by a microfluidic channel. A pulsatile flow is established through an on-chip micropump (adapted from Wu et al.49), enabling cross-talk between the organ equivalents.
Fabrication of the 2-OC was performed as described by Wagner et al.15. Briefly, replica moulding of polydimethyl-siloxane was applied in a 2-mm high layer containing the respective microfluidic channel, micropump and openings for culture compartments. It is sealed at the bottom by permanent bonding to a glass microscope slide (Menzel, Braunschweig, Germany) using low pressure plasma oxidation (Femto; Diener, Ebhausen, Germany), thus, forming a fluid-tight channel with a height of 100 µm.
The on-chip micropump was actuated by pressured air or vacuum, resulting in a consecutive lowering and raising of the 500-µm thick elastic membranes.
Differentiated HepaRGs (Lot HPR116080 or HPR116189) were obtained from Biopredic International (Rennes, France). Primary human hepatic stellate cells (HHSteC) were purchased from BioreclamationIVT (Westbury, NY, USA). The 3D pancreatic islet microtissues were purchased from InSphero (Schlieren, Switzerland)27.
The differentiated HepaRGs were thawed and seeded confluently four days before spheroid formation. Standard HepaRG culture medium consisted of William's Medium E (PAN-Biotech, Aidenbach, Germany) supplemented with 10% foetal calf serum (Corning, Lowell, MA, USA), 5 µg/ml human insulin (PAN-Biotech), 2 mM L-glutamine (Corning), 5 × 10−5 M hydrocortisone hemisuccinate (Sigma–Aldrich, St. Louis, MO, USA), 50 µg/ml Gentamycin Sulfate (Corning) and 0.25 µg/ml Amphotericin B (Corning) or was purchased directly from Biopredict. An amount of 0.5% dimethyl sulfoxide (Carl Roth GmbH, Karlsruhe, Germany) was added to the medium to keep the HepaRGs in a differentiated state. On the following day, the medium was renewed with HepaRG medium containing 2% dimethyl sulfoxide. The cells were maintained in this medium for three days until spheroid formation.
HHSteC (Lot PFP) were expanded in Stellate Cell Medium, provided by ScienCell (Carlsbad, CA, USA). Cells were used between passage 3 and 6. Pre-culture was started at least two days before spheroid formation.
Upon the arrival of pancreatic islet microtissues in 96-well GravityTRAPTM plates, the microtissues were treated according to the manufacturer's protocol and maintained for two to five days in human islet maintenance medium (InSphero) before transfer to the 2-OC.
Human liver spheroids were formed combining differentiated HepaRG cells and HHSteC using 384-well spheroid microplates (Corning) in HepaRG medium containing physiological amounts of insulin (1 nM). Briefly, 50 µl containing 24,000 hepatocytes and 1,000 HHSteC was pipetted into each well of the spheroid plate. The plate was centrifuged for 1 min at 300 g and incubated at 37 °C and 5% CO2. Compact spheroids formed within four days. Forty spheroids were collected in a 24-well ultra-low attachment plate (Corning) in HepaRG medium containing 1 nM insulin. The plate was placed on a 3D rotator (PS-M3D; Grant, Shepreth, UK) for one day before transfer to the culture compartments of the 2-OC.
Liver spheroids were collected for RNA isolation using the NucleoSpin RNA Kit (Macherey-Nagel, Berlin, Germany). cDNA was synthesized by reverse transcription of 200 ng total RNA using the TaqMan® Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, USA). Quantitative PCR was performed using the QuantStudio 5 Real-Time PCR System (ThermoFisher Scientific) and the SensiFAST SYBR Lo-ROX Kit (Bioline, Luckenwalde, Germany), according to the manufacturer's instructions. The real-time qPCR primers were as follows: insulin receptor forward 5′-CGAGAAACTGCATGGTCGCC-3′ and reverse 5′-CACGCCAAAGGACCACATGTC-3′ and TATA-box-binding protein forward 5′-CCTTGTGCTCACCCACCAAC-3′ and reverse 5′-TCGTCTTCCTGAATCCCTTTAGAATAG-3′.
HepaRG cells were seeded confluently (200,000 cells/cm2) in 12-well plates and maintained, as described previously, for eight days. Stellate cells were seeded at a density of 13,000 cells/cm2 in 12-well plates and maintained for four days. Liver spheroids were formed as described above. One day before performing the assay, liver spheroids and monolayer cultures were washed three times with phosphate buffered saline (PBS; Corning) and cultured in assay medium, consisting of Williams E (PAN-Biotech) with 5.5 mM glucose and 0.1% bovine serum albumin (Serva, Heidelberg, Germany), overnight. The following day, cultures were incubated in assay medium with 1 nM or without insulin (PAN-Biotech) for 30 minutes at 37 °C. Reactions were stopped by washing cultures three times with 1.5 ml ice-cold PBS on ice. Subsequently, the cells were lysed in 100 µl cell extraction buffer (FNN0011; Thermo Fisher Scientific) containing 1 mM PMSF protease inhibitor (Thermo Fisher Scientific) and protease inhibitor cocktail (P8340; Sigma-Aldrich) for 30 min (monolayer) or 60 min (spheroids). Cell extracts were snap frozen and centrifuged at 13,000 g for 10 min at 4 °C after thawing. Protein concentrations in supernatants were determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific). The ratios between phosphorylated and unphosphorylated AKT were quantified using a human AKT ELISA kit (KHO0111; Therma Fisher Scientific) and a human phospho-AKT pS473 ELISA kit (KHO0111; Thermo Fisher Scientific), as recommended by the manufacturer.
The 2-OC cultures were conducted in standard HepaRG culture medium without insulin, in short including William's Medium E (11 mM glucose), 10% foetal calf serum, 2 mM L-glutamine, 5 × 10−5 M hydrocortisone hemisuccinate, 50 µg/ml Gentamycin Sulfate and 0.25 µg/ml Amphotericin B. Prior to transfer, the organ equivalents were washed twice with PBS/0.1% BSA and equilibrated for 2 h in co-culture medium. Forty liver spheroids and ten pancreatic islet microtissues were loaded into the spatially separated culture compartments of a common circuit. Additional circuits were loaded with either 40 liver spheroids solely or 10 pancreatic islet microtissues solely, for comparison. Each culture compartment was filled with 300 µl medium. A complete medium exchange in both compartments was performed after 24 h of culture. Subsequently, the medium was renewed completely every 48 h. Cultures were ended after 15 days of culture and organ equivalents were collected from the culture compartments.
To perform an in vitro glucose tolerance test, medium from both culture compartments was completely removed and replaced with 315 µl medium, respectively, containing 11 mM glucose. Samples of 15 µl were collected immediately from both liver and islet compartments and pooled to obtain sufficient sample volume for the subsequent analyses. The procedure was repeated for sample analyses following 8, 24 and 48 h dynamic cultivation. This all leads to a maximum volume reduction of 10%, as a complete medium exchange was carried out directly after taking the sample at 48 h. The total area under the glucose and insulin curves was calculated using the trapezoidal method.
Eleven islet microtissues per condition (n = 2–4 islet microtissues per circuit) were removed from the culture compartment and collected in separate wells of a GravityTRAPTM plate (InSphero) to analyse the islet functionality by GSIS. After an initial equilibration for 2 h in low glucose buffer (2.8 mM), the islet microtissues were washed and sequentially incubated in 50 µl low glucose buffer (2.8 mM) and then high glucose buffer (16.8 mM) for 2 h, respectively. The buffers were collected after incubation and analysed using a human Insulin ELISA kit (10-1113-01; Mercodia, Uppsala, Sweden).
Medium samples were collected during a complete medium exchange every 48 h for glucose, insulin and albumin analyses. Additionally, samples were taken after 0, 8, 24 and 48 h on day 1, 7 and 13 to measure the response to a high glucose load.
Glucose concentrations were measured using the GLU 142 kit (Diaglobal, Berlin, Germany), according to the manufacturer's instructions, with minor modifications: 95 µl of the reagent was mixed with 5 µl of prediluted sample and incubated for 20 min at room temperature. Absorbance was measured at 520 nm using a standard curve prepared with Williams E containing 11 mM glucose as a reference.
Commercially available ELISA kits were used to measure the insulin (Mercodia) and albumin (E80-129; Bethyl Laboratories, Montgomery, Tx, USA) concentrations. Absorption was determined using a microplate reader at 450 nm. Albumin production was calculated by multiplying the albumin concentration with the remaining volume in the compartment after 48 h of culture.
Differences among groups were analysed using unpaired two-tailed Student's t test or ANOVA as appropriate, and a p value < 0.05 was considered statistically significant.
A correction to this article is available online at https://doi.org/10.1038/s41598-018-20340-1.
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We gratefully acknowledge Philip Saunders for critical editing of the article and Peter Mangel for the preeminent graphic visualisation of the 2-OC. Research reported in this publication was supported by the German Federal Ministry of Education and Research, Grant No. FKZ 01DJ14010A.
Uwe Marx and Tommy B. Andersson jointly supervised this work.
S.B.: designed and conducted experiments, reviewed and interpreted results and drafted portions of the manuscript. C.W.H.: designed and conducted experiments, reviewed and interpreted results and finalized the manuscript. K.K.: designed and conducted experiments, reviewed and interpreted results and finalized the manuscript. I.D.: conducted chip culture experiments, and cell culture media and endpoint analyses. D.G.: conducted chip culture experiments, and cell culture media and endpoint analyses. S.A.: developed the study concept, interpreted results, finalized the manuscript. L.E.: developed the study concept, interpreted results, finalized the manuscript. W.G.H: developed the study concept, interpreted results, finalized the manuscript. I.M.: designed and supervised experiments. A.W.: designed and supervised experiments. C.Ä.: designed and supervised experiments and finalized the manuscript. U.M.: developed the study concept, interpreted results and finalized the manuscript. T.B.A.: developed the study concept and study design, interpreted results, and drafted and finalized the manuscript.
Authors declare no competing financial interests. U.M. is a shareholder and CEO of TissUse GmbH, Germany, which is marketing the MOC platform.


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BGT dancer due to perform for first time after back operation funded by Simon Cowell
Pascale DayFriday 9 Feb 2018 1:25 pm
Sorry, this video isn't available any more.
Julia Carlile, a BGT contestant, is bouncing back and ready to perform after recovering from back surgery paid for by Simon Cowell.
The dancer, who is part of the dance group Mersey Girls, appeared on This Morning on Friday to show off the results of her painful operations to help with her scoliosis.
The 15-year-old and her four teammates cried on the morning show after a VT of her journey was played out, as Ruth Langsford handed tissues out to the emotional teens.
Julia spoke about her journey of recovery, saying that her first time back dancing showed her that she had completely 'lost confidence'.
The Mersey Girls are finally able to perform as a fivesome again (Picture: ITV)
'When I started I figured out I'd lost strength, flexibility and confidence, because I was terrified to do stuff.'
She revealed her affliction on Britain's Got Talent earlier in 2017, and Simon Cowell immediately stepped in and offered to pay for the operation.
Available only in the US, the expensive surgery involved using a flexible chord to correct the spine rather than the usual rods, which would allow her to continue working towards a career in dancing.
Julia received the first operation on July 25, and she's since had the second, both lasting six hours each.
Julia has done it! First op done, we are so proud of her and missing her so much want to give her a MASSIVE HUG, our girl is a fighter❤❤ pic.twitter.com/9mjOXrlrSd
— Mersey Girls (@MerseyGirls) July 25, 2017
In a previous interview with This Morning she had called the operations 'hardest thing I've ever done,' but Julia wasn't one to give up, with her mate and fellow Mersey Girls member Annie saying about her recovery: 'Even when we were in America she was stretching and I was like Julia, please stop! But she doesn't stop.'
The dance troup are performing for the first time since Julia's surgeries at Britain's Got Talent's Big Celebration in Liverpool at the weekend.
'It's basically the first time I'm dancing again in public since my surgery,' she said. 'So that's exciting!'
This Morning continues weekdays from 10.30 on ITV1.
If you've got a celebrity story, video or pictures get in touch with the Metro.co.uk entertainment team by emailing us celebtips@metro.co.uk, calling 020 3615 2145 or by visiting our Submit Stuff page - we'd love to hear from you.
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redpajama_set_name: RedPajamaCommonCrawl


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Since 1 January 2019, SIEBTECHNIK TEMA has been the umbrella brand of SIEBTECHNIK GmbH, founded in 1922, and of several companies of TEMA Holding N.V., founded 1925.
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Polish Airlines LOT brings back Budapest-New York flight
Budapest Airport confirmed that LOT is bringing back its Budapest-New York flight from June 2022.
Polish Airlines LOT is bringing back its Budapest-New York flight from June 2022. Budapest Airport said the route, initially launched in the spring of 2018, was temporarily suspended because of the coronavirus pandemic. LOT will fly Boeing 787 Dreamliner aircraft on the route which will operate three times a week.


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Paul scores 41 as Rockets eliminate Jazz 112-102
Chris Paul scored a playoff career-high 41 points, helping the Houston Rockets eliminate the Utah Jazz 112-102 in Game 5 on Tuesday night.
Paul scores 41 as Rockets eliminate Jazz 112-102 Chris Paul scored a playoff career-high 41 points, helping the Houston Rockets eliminate the Utah Jazz 112-102 in Game 5 on Tuesday night. Check out this story on thespectrum.com: http://www.thespectrum.com/story/sports/2018/05/08/paul-scores-41-rockets-eliminate-jazz-112-102/593284002/
Associated Press Published 9:07 p.m. MT May 8, 2018 | Updated 10:33 p.m. MT May 8, 2018
Utah Jazz guard Donovan Mitchell, center, passes the ball as Houston Rockets forward Trevor Ariza, left, and center Clint Capela defend during the first half in Game 5 of an NBA basketball second-round playoff series, Tuesday, May 8, 2018, in Houston. (AP Photo/Eric Christian Smith)(Photo: The Associated Press)
HOUSTON — Chris Paul scored a playoff career-high 41 points with eight 3-pointers to finally reach the conference finals, helping the Houston Rockets eliminate the Utah Jazz 112-102 in Game 5 on Tuesday night.
It will be Houston's second trip to the Western Conference finals in four years and the first ever for Paul. The point guard has been panned for failing to get past the second round in his nine previous trips to the postseason.
Houston will meet the winner of the series between Golden State and New Orleans, which the Warriors lead 3-1.
Paul, a nine-time All-Star in his first season with Houston after an offseason trade from the Los Angeles Clippers, also had 10 assists and seven rebounds. His previous career-best in the playoffs was 35 points, which he accomplished three times.
Star rookie Donovan Mitchell had 22 of his 24 points for Utah in the third quarter before leaving with about seven minutes left with an apparent left leg injury.
Houston Rockets guard Chris Paul, right, watches from the sidelines during the first half in Game 5 of an NBA basketball second-round playoff series against the Utah Jazz, Tuesday, May 8, 2018, in Houston. (AP Photo/Eric Christian Smith) (Photo: Eric Christian Smith, AP)
Houston returns to the conference finals for the first time since 2015. It's the second straight season the Jazz lost in this round after being swept by Golden State last season.
The top-seeded Rockets led by eight points after a 3-pointer by Paul with about six minutes left. The Jazz scored the next seven points, highlighted by a 3-pointer from Royce O'Neale, before Paul made eight quick points to push Houston's lead to 105-96 with about three minutes left.
Joe Ingles made a basket for Utah before Paul added another basket to make it 107-98. The crowd began chanting "CP3" repeatedly as Paul dribbled down the court on the next possession.
Houston Rockets guard James Harden (13) is fouled by Utah Jazz forward Jae Crowder (99) during the first half in Game 5 of an NBA basketball second-round playoff series, Tuesday, May 8, 2018, in Houston. (AP Photo/Eric Christian Smith) (Photo: The Associated Press)
The Jazz were without Ricky Rubio and Dante Exum because of hamstring injuries. Rubio has missed the entire series with his injury, and Exum was injured in the third quarter of Game 5.
P.J. Tucker scored a playoff career-best 19 points, and James Harden added 18 points.
Mitchell's huge third quarter left the Jazz up by 3 to begin the fourth. Derrick Favors made a layup for Utah to start the final quarter before Paul and P.J. Tucker hit consecutive 3-pointers to put Houston back on top 81-80 with about 10 minutes left.
Utah regained the lead on a basket by O'Neale before Houston used an 11-5 spurt, with the first five points from Gerald Green, to take a 92-87 lead with about seven minutes left. Harden capped the run with a dunk after stealing the ball from Mitchell. Mitchell was injured on that play and grabbed his left leg. He had to be helped to the bench and was quickly taken to the locker room with the help of two people.
Jazz: Alec Burks scored 22 points off the bench. ... Rudy Gobert finished with 12 points, nine rebounds and five blocks. ... Mitchell finished 9 of 17 on field goals. ... O'Neale had 17 points.
Rockets: Houston made 18 3-pointers to give the team 10 or more 3-pointers in an NBA playoff-record 16 straight games. ... Coach Mike D'Antoni turned 67 on Tuesday. ... Clint Capela finished with five blocks.
Last week's Athlete of the Week propelled PVHS
Popular St. George bowler keeps knocking 'em down
Mucho Gusto romps in Pegasus World Cup
Mesquite bowler rolls way to a career night
DH preps for Cedar rematch, PV gets Jensen back
Analyzing the RPI, and the top 3 wins so far
Dixie State
Mesquite E-Newspaper
© 2020 The Spectrum & Daily News, a division of Gannett Company, Inc.
Cars.com St. George


Familiarize yourself with the 2014 INFINITI QX60. It features a continuously variable transmission, all-wheel drive, and a 3.5 liter 6 cylinder engine. INFINITI infused the interior with top shelf amenities, such as: front and rear reading lights, power front seats, a trip computer, telescoping steering wheel, a power liftgate, remote keyless entry, and more. Passengers in the third row enjoy seat back reclining functionality, providing an extra level of comfort and convenience. With high intensity discharge headlights illuminating your path, you'll always appreciate maximum visibility. For drivers who enjoy the natural environment, a power moon roof allows an infusion of fresh air. INFINITI ensures the safety and security of its passengers with equipment such as: dual front impact airbags with occupant sensing airbag, front side impact airbags, traction control, brake assist, a security system, and 4 wheel disc brakes with ABS. Various mechanical systems are monitored by electronic stability control, keeping you on your intended path. We have a skilled and knowledgeable sales staff with many years of experience satisfying our customers needs. We'd be happy to answer any questions that you may have. Come on in and take a test drive!


text:
package ru.generator;

import ru.generator.Parsers.TsvPars;
import ru.generator.Parsers.XmlParameters;
import ru.generator.out.WriteInFile;

/**
 * Created by Anton on 11.07.2017.
 *
 */
public class Generator {

    /**
     * Start.
     * @param args paths
     */
    public static void main(String[] args) {
        Generator generator = new Generator();
        generator.start(args[0], args[1], args[2]);
    }

    /**
     * Start Generator.
     * @param path1 xml file
     * @param path2 tsv file
     * @param path3 txt file
     */
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        try (WriteInFile writeInFile = new WriteInFile(path3)) {

            XmlParameters xmlParameters = new XmlParameters(path1);
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                formatterTable.addRow(formatterRow.formatter(tsvPars.getDataNext()));
            }
        } catch (Exception e) {
            e.printStackTrace();
        }
    }
}

redpajama_set_name: RedPajamaGithub


text:
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text:
All women have Draupadi in them: South African director
New Delhi, March 7 (Calcutta Tube) South African director Tina Johnson, who is directing a modern-day take on Draupadi of the Hindu epic 'Mahabharata', says she sees the queen of the five Pandava brothers as a Greek heroine. She adds that all women 'have a bit of Draupadi in them'.
'She is a Greek heroine who suffers tragedy and comes out stronger. She is angry, humiliated and is an archetypal wronged woman seeking justice,' Johnson, who is directing the Indian play 'Draupadi: We are no different now', told IANS.
The play is being produced by Odissi dancer, actress and compere Shivani Wazir Pasrich and the weeklong production will be staged March 19-26 at the Kamani Auditorium here.
The play comprising a cast of seven is a modern-day adaptation of Draupadi's episodes in the 'Mahabharata'. The epic heroine is portrayed in two avatars: a young woman struck by personal grief and an aging matron.
Draupadi, feels Johnson, shares more similarities with 'South African women than American women'.
'But all women have a bit of Draupadi in them. It is complicated experiment. We are trying to create a character who is humiliated in modern times – juxtaposed against the ancient Draupadi. There are 15 stories woven around around two women,' said Johnson.
The play, says Johnson, centres around Maaya.
'The young woman comes to know that her fiance, a sculptor, is burnt to death. She tries to kill herself, but an older woman Draupadi stops her,' she says.
In return, Draupadi demands a favour. Maaya wavers and is unable to deliver. Soon the events in Maaya's life shows 'an uncanny resemblance to the events in Draupadi's life'.
This is not Johnson's first 'woman-oriented' play.
'I had directed a play on a community in Cape Town known as the 'coloured community' comprising Portuguese, African and White people. The play, 'A Coloured Place', was about women. But Draupadi has a different context,' she said.
Tina Johnson has worked in New York City, Johannesburg and Cape Town at the Market Theatre and the Baxter Theatre. Her plays include 'Hamlet', 'Lysistrata', the 'Coolie Odyssey', 'Itsoseng' and 'Out of Bounds'.
'Itsoseng' and 'Out of Bounds' topped the jury's list at the Edinburg Theatre Festival. She has trained actors, directors and worked as mentor for university students at the Grahamstown National Arts Festival and served as the deputy chair at the Gauteng Theatre Practitioners.
The director feels interpreting Draupadi is 'difficult because of the complexities of the emotions involved'.
'I have read the 'Mahabharata'…though mostly the children's version in English…,' she said.
Johnson's 'Draupadi…' has an impressive 'behind-the-scene' crew.
Veteran artist Anjolie Ela Menon has conceived the artistic backdrop of the play, which includes the two faces of Draupadi that serve as the artistic leitmotifs on the T-shirts and the layered clothes that ace designer Ritu Kumar is creating for the cast. While one of the faces is dark and poignant, the other is young and innocent.
Shubha Mudgal is setting the score and period designer Aman Nath is creating the heritage stage.
'It will be an intimate one-and-a-half hour drama heavy with dialogues. But the drift is modern and characters speak an everyday language – like the way we speak. The play can be best described as revenge versus resilience,' Shivani Wazir Pasrich, who plays one of the leading ladies, told IANS.
Pasrich had co-produced the grand opera, 'Karna' with Farrukh Dhondy with Varsha Bedi in 2008.
(Madhusree Chatterjee can be contacted at madhu.c@ians.in)
Bengali Little magazine 'Shabda Harin' promotes newcomers
By Shrabanti Basu / March 5, 2012
Rabindranath Tagore's songs in English translation
By Bengali Movie / March 3, 2012
Tagore art and sculpture exhibition in Kolkata
By Calcutta Tube Team / March 3, 2012
Kolkata hosts Tagore children's film festival
Mumbai to host Bengali Music Fest
By Calcutta Tube Team / February 8, 2012
Egyptian Sufi dance sets the capital alight
Malini Ramani takes audience on martini trip at Lakme Fashion Week


We've all heard it said that kitchens are the heart of the house, and given the time spent in most kitchens, it is no surprise that this is the place where most homeowners will spend the most money.
The kitchen, in many ways, is the most complicated room in a home because it needs to accommodate more functions than any other space. When embarking on a custom kitchen design, the list of considerations is immense. An effective way to organize the process of design is to categorize the details under the headings of: use, layout, style, budget and materials.
This column marks the first of a two-part column that will first talk about use and layout, and then address style, budget and materials in the next article.
Determining how a kitchen is used lends itself to the exploration of the habits, hobbies and day to day functions of your household. Do you love to bake or have elaborate dinner parties? Or do you have small children and need a space to watch over them as they do their homework while you prepare dinner?
Mapping out the way you plan to use your space will help you figure out which appliances you need, what kind of work space may be required, and even how the space needs to flow in order to carry out the various tasks that need to be accomplished on a regular basis. The end of this initial exploration should bring about a wish-list of components that establishes the program for your kitchen. This program can then be used to layout your space.
Kitchen layouts can be as unique as the families they are designed for, but there are fundamental principles that can be adapted to organize an efficient space. Most kitchen planners will start with a workspace "triangle" composed of a sink, fridge and cook top, or range. You typically don't want to have to travel more than three to seven feet within the confines of this triangle, but with the advent of even more appliances like built-in espresso machines, steam ovens, microwaves and refrigeration systems that separate fridges from freezers, the traditional "triangle" is starting to look like a crazy many sided polygon. All these new appliances, combined with fixtures like bar sinks, ice-makers and hot water dispensers can certainly leave one's head spinning when searching for an organizational strategy.
One way to start would be to move from specific to general layout planning. In order to do this, you can think of breaking down each activity. For example, let's think of what it takes to get you your first cup of morning coffee. If you have a built-in coffee machine, it might be good to have it located by the china cupboard for mugs, near a sink for accessing water or cleaning parts, and by the fridge for cream. Thinking about how you use each appliance starts to influence what surrounds them, and the relationships they have with each other. In essence, the various operations of each appliance and fixture will actually start to dictate its location.
This matrix of kitchen tools will then be grounded by circulation, work stations, and places of repose. Some of the more typical layouts or shapes for kitchens are the U-shape, L-shape, or a galley style kitchen. These more traditional shapes assume only one work station in the kitchen, but the evolution of design ideas along with the growing sizes of kitchens and lifestyle changes have created even more shapes like a double L that incorporates a peninsula, or even a G shape that has a free-standing island in the middle of a U-shape kitchen.
Regardless of the shape of the kitchen, circulation paths should not be less than three feet or more than six feet with work areas set apart so as to not interrupt food or beverage preparation. The same can be said of the dining areas in a kitchen like a raised bar, or nook area. It is, however, important that these spaces maintain not only a visual connection to the work areas, but be able to interact with them as well.
With today's busy schedules, having places in the kitchen where guests and family members can interact with those preparing meals has become a critical component in design. More often than not, meal preparation is just as much of a social activity as the actual dining.
Today's kitchen is not only the source of physical sustenance, but also a place that facilitates the mingling of friends and family. In this sense, the kitchen is, truly, the heart of the home.
With the unsettling realities of the state of the world settling into 2009, the idea of having a place to decompress or, quite frankly, hide is a welcome notion.
I cannot count how many times clients have come to us with the request of wanting to have a bathroom with a "spa-like" quality—a place where they can have a temporary reprieve from the comings and goings of the day-to-day grind. If you really think about it, this is a pretty tall order for a single room in a house to fill, but it is amazing how having just one space dedicated to being a sanctuary can influence the overall feeling in the rest of the home. The way a bathroom is equipped, designed and detailed can certainly enhance this experience.
On the most basic level, the bathroom is conceived of as a purely functional space traditionally associated with sanitizing and often times, rushing around. However, the need for a place for stillness has inspired a series of new concepts to help outfit today's bathrooms. Each concept, infused with technology and design, isolates each activity to a separate zone.
The tub area should be situated in an area that has the potential to evoke the most serenity. It should be furthest away from the door, and off the path of circulation. Placing a tub in an alcove, with an operable window to welcome in fresh air or glimpses to a peaceful outdoor scene can certainly enhance the bathing experience. It is also a great idea to have a wide enough tub deck to allow for the placement of candles, or even a glass of wine. Tubs themselves have come a long way and are not only available in a variety of sizes and shapes, but may also have the added soothing benefits of air jets that create the effect of bathing in champagne bubbles, or water jets that can massage tired muscles. One of the newest features available in tubs is chromatherapy. Chromatherapy uses rhythmic coloured light sequences that are believed to aid in the restoration of inner balance. In the end, however, sometimes nothing beats just a good, hot soak in a traditional clawfoot tub.
The idea of taking a "quick shower" can also surrender to the notion of infusing more relaxation into one's daily cleansing ritual. Today's showers have the option to come equipped with a "shower system." A combination of multiple sprays, or a shower arm combined with a large rain shower head, or waterfall spout can facilitate a varied and soothing experience. Steam showers are also quite popular, but they need to be installed with benches, and proper ventilation in order to have the full effect of an in-home sauna. It is also nice, space permitting, to have a shower stall that is designed for two.
Unlike showers, tubs, or toilets for that matter, the wide selection of sinks and faucets available has little to do with technology and almost everything to do with esthetics. In this sense, the design of the vanity area provides an opportunity for a unique architectural expression. In addition to selecting fixtures and finishes that best suit the overall feeling you are trying to evoke, consider having varied counter heights to accommodate different functions. A lowered area, between 27 and 29 inches, is a perfect height for applying makeup while sitting down, while a sink height of 32-34 inches is best for tasks that require standing up. You can even incorporate counter heights that range from 36-42 inches to facilitate more storage.
These cleansing stations seem best served when there is some separation from the toilet area. This can be achieved by either creating a separate room, or by erecting a small wall to block the sight line. However, this most base of all bathroom activities need not be bereft of luxury. Toilet technology aside, natural lighting, good ventilation, and beautiful finishes can make the toilet area a part of the spa experience.
- Provide a mixture of natural and artificial lighting that can be adjusted to create a serene setting.
- Install a heated floor.
- Provide plenty of ventilation, natural and mechanical.
- Make certain there is plenty of storage and areas to hang towels and robes.
Unlike most rooms in a home, the bathroom is perhaps the only room where it is perfectly acceptable to just lock the door and, on a more symbolic level, shut out the day's worries. In this sense, it is the perfect place to create a private oasis that offers a peaceful and invigorating respite from the rest of the world.
One of the first things that should be done when considering embarking on any type of construction project that may add square footage to, or change the exterior look of your home is a zoning analysis.
More often than not, a designer can help you realize your design goals while working within the confines of your zoning. That said, there are times when zoning bylaws, be they outdated, or somewhat vague, inappropriate, or seemingly unfair when measured up to your project, can be challenged. The arena for this bold act is known as a board of variance hearing.
All municipalities have different schedules and different procedures for processing appeals of this nature; however, there are some general guidelines and tools you can arm yourself with if you choose to go down this path. My design partner, Kevin Vallely, and I were recently successful at a hearing in the City of Vancouver in which all the terms of our appeal were granted. Included in our appeal was a request for a relaxation in height, and allowable square footage. Although the actual presentation and verdict took all of 15 minutes, our process for this appeal began in early June.
We had already been working with our clients for a couple of months and had reached a point in the design process where all parties were happy with the direction the design was heading. Having done technical checks throughout our process, we were aware of the non-conformities we were facing and had started to initiate discussions with the planning department in order to confirm our findings and get a sense of whether what we were asking for was reasonable. Depending on the complexity of your appeal, it is often a good idea to establish a history/relationship with the planning department so that when you present, they are familiar with your case. They can even offer some valuable suggestions and advice on how to navigate through this process. One has to note that the local authority is assuming a great deal of responsibility for allowing the construction of what you may be proposing and will therefore not take requests lightly. And although the planning department cannot actually support any attempts to effectively challenge the bylaws, they can certainly let you know that they will not "oppose" your appeal.
In order to have any success with the board of variance, you need to have a strong case. What they are typically looking for are sound reasons for why you think they should relax their regulations. The foundation for these "reasons" usually rests in a situation known as a "hardship." Hardships arise out of a variety of incidences. In our instance, we were trying to work with an existing structure built in the 1940s. As a result, the basement elevation was much higher than where one would set it today in order to have it not count in the total allowable square footage. This higher basement also made it so that adding a second storey would easily set it beyond allowable height. In this sense, our arguable hardships would be trying to work with an existing condition while respecting a budget that simply could not afford to tear down the house and build new.
Once the base for your argument is established, you need to provide supporting documents that demonstrate that you have done your homework and are prepared to argue your case. Different boards will require different documents, but most will look for preliminary construction documents, letters describing your appeal, and photos of the existing area with your proposal somehow worked into those images. At this stage of the game, you really need to think about your submission package and whether its contents make for a compelling presentation, and if it can defend you against any opposition either from the members of the board, or a curious neighbor. The more prepared you are, the better. Having a three-dimensional model, or a beautiful rendering will only benefit your presentation. You can use these tools to demonstrate that your project will not have a negative impact on your neighborhood and may in fact enhance its overall appeal. There are also times when it is a great idea to canvas your neighbors and potentially get them to sign letters of support for your project.
Taking all these steps may not guarantee success, but being prepared will greatly increase your chances. There are always risks when you challenge authority. In the case of an appeal to the board of variance, you will have to be prepared to spend the extra time and money required to pull together a decent presentation, but a successful appeal is an amazing thing when you get the green-light to move forward and become a few steps closer to realizing your construction goals.
In the last article, we looked at how determining the specific uses in a kitchen can help inform its layout with regards to circulation, work stations and areas of repose.
While one starts to organize these components, thoughts should simultaneously be given to style, budget and materials.
A great place to start when attempting to establish the style of the kitchen is magazines. By flipping through magazines and cutting out pictures of things that you like, you are effectively creating your own "style journal." A designer can then take your collection of images, extract your ideas and begin to create a coherent vision that incorporates the essence of what you are looking for. Establishing the theme of a kitchen, be it modern, traditional or contemporary, will help guide you through the task of selecting materials, appliances, plumbing and lighting fixtures.
A modern kitchen is typified by clean, simple lines with close attention given to the concealment or strict organization of kitchen tools and culinary accoutrements. Surface materials are often more uniform, favouring edge grain woods and stone surfaces with less movement or texture. Not all modern-styled kitchens have to have the requisite stainless steel appliances, but if you think of the basic attributes of steel—easy to clean, smooth and uniform sheen—it makes a lot of sense. Modern does not necessarily have to be stark, and there is nothing preventing you from incorporating hits of colour and texture through some carefully selected accessories and textiles.
On the other end of the spectrum, a traditionally styled kitchen may draw on inspiration from early French, English or Italian styling. Cabinet doors with raised panels made of rich, deeply coloured woods or antiqued finishes, thick clay tiles, or stone with a tumbled or marbled character start to form the base of the esthetic. One may also consider incorporating classically patterned tile inserts, distressed woods, and crown mouldings complete with baseboards and trims. A traditional kitchen usually has a heavier feel than a modern kitchen since it is often grounded by detailed flourishes and embellishments.
A contemporary kitchen is a little more difficult to define as it seems to draw from a mixture of styles. Currently, it seems that we have reached a point in design where seemingly anything goes. This eclecticism is not really a new idea except in the formal pinning of it as a term used to define style. The truth is, most of us decorate our homes in this way: we collect and incorporate things we like.
All this said, it doesn't hurt to exercise a bit of discipline in order to maintain a sense of cohesion. In this sense, having a consistent theme will help unify your space. This is where using similar colours and materials can help pull things together.
The finishes you choose play a critical role in further defining the type of space you are trying to create. The combination of flooring, backsplashes, cabinets, countertops and hardware will effectively become your kitchen palette. At this point of the design process, it is a good idea to visit the various showrooms that showcase these items and pull together a selection of things that appeal to you. You may discover that you love the idea of using bamboo as your flooring, or that you simply have to have a tile mosaic backsplash bursting with colour.
Working with a designer may reduce the legwork of travelling from showroom to showroom since he/she may have access to a variety of samples on hand; however, I think that these visits help provide an excellent injection of inspiration. Once you have gathered a collection of samples, you can then lay them out on a table and play with different combinations by switching out one material for another until you are satisfied with the overall composition.
When selecting materials, fixtures and appliances you should never lose sight of the budget. This is where having a clearly established vision will help in guiding your choices.
Some budgetary strategies may include exploring the use of laminates over hardwoods and stone, visiting auctions or checking the paper for appliance or cabinetry sales, and using stocked cabinets in lieu of custom millwork. A stylish kitchen doesn't need to break the bank.
Even though a budget may determine the level of finish, it doesn't need to override stylistic intentions if you are prepared to make well considered compromises while working within your chosen theme.
DESIGNSTUDIO8 is a design collaborative providing thoughtful modern design. We seek to uncover unique design solutions that are innovative in their response to client's needs, site opportunities and budget. We have a hands-on approach and look at design holistically. Our work is inherently contemporary and sustainable, and each project is as individual as its client. Our projects have been recognized as Green Build Award Winners and Georgie Award Winners.
Our focus is on working with you to transform your ideas into a creative, inspiring and poetic space. We find pleasure in simple things like in the way people inhabit spaces and how a daily routine can be supported by design. Great teams make great projects. We include consultants and contractors in each phase of design, to create a synergy that allows for smooth coordination through construction. We draw from our broad experience working internationally and locally in West Vancouver.
We like to share our enthusiasm and drive to take an idea and help it to grow into something amazing! If you would like a guided tour through any of our projects, let us know and we can coordinate.
Lucia brings an extensive residential and interior design background to each project. She has sixteen years of experience on medium- to high-end projects and has managed over 60+ residential projects at DS8 and Kallweit Graham Architecture. She spent 4 years working abroad in New York and San Francisco where she gained invaluable experience working on a wide range of commercial, retail, hospitality and mixed used projects. Lucia has also developed skills in landscape design and has published articles in the "North Shore News" as part of the "Building by Design" series, providing design insight to home owners.
Tina brings a multi-disciplinary approach to her design work drawing from her background as an engineer. Her design work encompasses innovative technologies such as rammed earth, inflatable roofing systems, and material proto-typing with CNC machines. She has fifteen years of diverse experience at firms such as PUBLIC, Hotson Bakker Boniface Haden Architects (now called DIALOG) and Lang Wilson LWPAC, working across Canada and London, UK. Her projects include large and small scale construction and renovation work in residential, commercial and institutional building types. She is a LEED Accredited Professional (Leadership in Energy and Environmental Design).
If you require a registered architect for your project, please contact Tina at Tina Hubert Architect at 604-720-8873.