Controle quantitativo e temporal de oxigênio Microenvironment no Ilhéu Individual
Summary
Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.
Abstract
Oxigenação simultânea e monitoramento de fatores de glicose no acoplamento estímulo-secreção em uma única técnica é fundamental para a modelagem de estados fisiopatológicos da ilhota hipóxia, especialmente em ambientes de transplantes. Técnicas de câmara de hipóxia padrão não pode modular os estímulos, ao mesmo tempo, nem fornecer monitoramento em tempo real dos fatores de glicose no acoplamento estímulo-secreção. Para superar estas dificuldades, aplicamos uma técnica de micro várias camadas de integrar tanto aquosa e modulações de fase gasosa através de uma membrana de difusão. Isto cria uma sanduíche estimulação em torno das ilhotas microscaled dentro do polidimetilsiloxano transparente dispositivo (PDMS), que permita o controlo dos factores de acoplamento acima mencionadas, através de microscopia de fluorescência. Além disso, a entrada de gás é controlado por um par de microdispensers, proporcionando, modulações quantitativos sub-hora de oxigénio entre 0-21%. Esta hipóxia intermitente é aplicada para investigar um novo fenômeno da ilhat pré-condicionamento. Além disso, armado com microscopia multimodal, fomos capazes de olhar para o cálcio detalhada e dinâmica do canal K ATP durante esses eventos hipóxicos. Nós encaramos hipóxia microfluídica, especialmente a técnica de dupla fase simultânea, como uma valiosa ferramenta no estudo de ilhotas, assim como muitos ex vivo tecidos.
Introduction
Dynamic hypoxia is important in biology, specifically for islet transplants
Dynamic hypoxia is an important physiological as well as pathophysiological parameter in many biological tissues. Change in oxygen, for example, is a potent developmental signal in angiogenesis. Moreover, spatial and temporal patterns in hypoxia modulate HIF1-alpha and play roles in diseases like pancreatic cancer. Hypoxia is also a confounding factor affecting islet transplant outcomes. Recently, temporally oscillations of hypoxia, or intermittent hypoxia (IH) have demonstrated benefits in "preconditioning" islets1. However, both static and transient hypoxia effects on islet physiology have yet to be well understood or studied, primarily due to the lack of appropriate tools to control islet's microenvironment.
Islets are well vascularized in vivo
Pancreatic islets are 50-400 μm spheroidal aggregates of endocrine cells, including beta-cells and alpha-cells that are responsible for glucose homeostasis. When islets are exposed to stimulatory glucose in the blood, uptake and glycolysis lead to ATP production, which opens up ATP-sensitive potassium (KATP) channels and results in calcium influx that triggers the exocytosis of insulin granules. Oxygen is important to drive this heavily metabolic process and insulin secretion is significantly influenced by the dynamics of blood flow and oxygen supply in addition to glucose gradients. Islets readily perform this glucose-insulin response in vivo as they are highly perfused in the pancreas, each within one cell length from a capillary vessel. However, the dense network of intraislet capillaries is removed by collagenase during islet isolation2,3. Consequently, both oxygen and nutrient supplies are constrained to a 100 μm perimeter due to diffusion limitations.
Current techniques have limited success in recreating islet microenvironment
Recreating islet's native oxygen and glucose dynamics, key to modeling physiological and pathophysiological conditions, is difficult to achieve with standard hypoxic chambers that require elaborate flow and lack continuous monitoring of islet functions. Moreover, transplant therapies for Type I diabetes expose isolated islets to hypoxia in the hepatic portal system4 which has much lower pO2 (<2%, 5-15 mmHg) compared to physiological pancreas (5.6%, 40 mmHg). Post-transplant, the islet grafts take two weeks or more to be revascularized. It has been demonstrated that hypoxic exposure impairs islet's glucose-insulin coupling mechanism. Among the stimulus-secretion coupling factors, calcium signaling, mitochondrial potentials, and insulin kinetics can be easily monitored using microfluidics. Our previous microfluidic technique demonstrated this real-time monitoring with precise modulation of the aqueous microenvironment around single islet5,6. However, quantification of islet's hypoxic impairment is stymied by the lack of simultaneous stimulation and monitoring techniques. Therefore, combining microfluidic control of oxygen and islet monitoring can improve islet hypoxia studies.
Microfluidics can recreate and modulate the aqueous and oxygen microenvironment
The standard technique for tissue and culture hypoxia studies has been based on hypoxic chambers. In general, the hypoxic chambers provide single oxygen concentrations with equilibration times in ~10-30 min, incompatible with minute-scaled dynamic hypoxia. Two recent studies used small custom chambers for intermittent hypoxia exposure on whole mice, with conflicting results on glucose-induced insulin response7,8. Bear in mind that at the whole animal level, the respired oxygen is not directly translated to islet capillary pO2, due to controls in the respiratory system. Furthermore, these studies do not have standardized oxygen levels, nor do they provide real-time measures at the tissue level of islets.
On the other hand, oxygen microfluidics can surpass these limitations by controlling oxygen via gas channel networks. Moreover, microfluidics is compatible with live imaging during oxygen modulation, a feat currently not possible with standard hypoxic chambers. A number of these novel microfluidics approaches utilize the gas permeability of polydimethylsiloxane to dissolve oxygen concentrations into microchannels that flow media over target cells9-14. These devices have also integrated multiple discrete oxygen concentrations, fluorescence based oxygen sensors, and even chemical oxygen generation on-chip.
Liquid solvation-based microfluidics have a hard time maintaining stable, continuous gradients as it depends on convective mixing which is sensitive to flow conditions. In comparison, the technique we use here focuses on decreasing the diffusion path of oxygen delivery. The gas solvation and shear flow are eliminated by directly diffusing oxygen across a membrane seeded with cells or islet tissues. This removes the extra microfluidics required to control solvation and prevents unnecessary shear stress to the islets, which itself can trigger insulin release. This platform has been used to demonstrate reactive oxygen species (ROS) up-regulation at both hyperoxic and hypoxic extremes (2-97% O2) in cell culture1,15. Because of the direct delivery of oxygen and removal of shear flow, our diffusion-based platform provides the optimal microfluidic solution for studying islet hypoxia.
Multimodal stimulation and monitoring
Diffusion-based microfluidics also brings additional benefits when adapted for studying islet microphysiology. By using a membrane as a diffusion barrier, the liquid can be isolated from the oxygen modulations, enabling controls of aqueous glucose stimulations independently from hypoxic stimulations. This creates a sandwich-like simultaneous stimulation that spatially pin-points delivery to the islets. Furthermore, as the gas is temporally modulated via computerized microinjectors, we can modulate the oxygen concentration from 21-0% digitally with transient time less than 60 sec. The dynamic controls of the oxygen and glucose microenvironment at the microscope allow a real-time multimodal protocol that would not be possible or extraordinarily cumbersome using standard hypoxic chambers. Using this device, calcium signaling (Fura-AM), mitochondrial potentials (Rhodamine 123), and insulin kinetics (ELISA) were monitored to provide a complete picture of the dynamic glucose-insulin response under hypoxia.
Protocol
Representative Results
Discussion
The multiple modalities integrated in this islet hypoxia technique present several points noted here for troubleshooting. First the isolated islets continue to degrade and disintegrate in culture due to digestive enzymes from acinar cells. Standardizing experiments to 1-2 days after islet isolation is thus critical in obtaining consistent results. Second, the aqueous flow was stopped during hypoxia and intermittent hypoxia to prevent convective clearance at the boundary between laminar flow and diffusion. This seems to l…
Declarações
The authors have nothing to disclose.
Acknowledgements
This work was supported by the National Institutes of Health Grants R01 DK091526 (JO), NSF 0852416(DTE), and Chicago Diabetes Project.
Materials
| Reagent/Material | |||
| Spinner | Laurell | WS-400 | |
| SU8 | MicroChem | SU8-2150/SU8-2100 | |
| Digital Hotplate | PMC Dataplate | 722A | |
| UV Curing Lamp | OmniCure | S1000 | |
| PMDS | Dow Chemical | Sylgard 184 | |
| Corona Wand | ETP | BD-20AC | |
| Vacuum Chamber | Bel-Art | 420220000 | |
| Microdispensers | The Lee Company | IKTX0322000A | |
| 5 V and 20 V DC Power | Radio Shack | ||
| NI USB | National Instrument | NI USB-6501 | |
| Thermometer | Omega Engineering, Inc. | ||
| Peristaltic Pump | Gilson | Minipulse 2 | |
| Oxygen Sensor | Ocean Optics | NeoFox | |
| Fraction Collector | Gilson | 203 | |
| Pippette | Fisher Scientific | Finnpipette II 100μl | |
| Inverted Epifluorescence Microscope | Leica | DMI 4000B | |
| 50 ml Conical Tubes | Fisher Scientific | ||
| Fura-2 Fluorescence Dye | Molecular Probes, Life Technologies | ||
| Rhodamine 123 Fluorescence Dye | Molecular Probes, Life Technologies | ||
| Culture Media | Sigma-Aldrich | RPMI-1640 | |
| HEPES | Sigma-Aldrich | ||
| Glucose | Sigma-Aldrich | ||
| Bovine Serum Albumin | Sigma-Aldrich | ||
| 30 in Silicone Tubings | Cole-Parmer | 1/16 in x 1/8 in | |
| 1.5 ml Eppendorf Tubes | Fisher Scientific | ||
| Y-connectors | Cole-Parmer | 1/16 in and 4 mm | |
| Syringe Connectors | Cole-Parmer | female Luer plug 1/16 in | |
| Straight Connectors | Cole-Parmer | 1/16 in | |
| Elbow Connector | Cole-Parmer | 1/16 in |
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