इमेजिंग न्यूरॉन Microfluidic संस्कृति प्लेटफार्म में Glia सहभागिता (MCP) आधारित Neuronal Axon और Glia प्रणाली सह संस्कृति विश्लेषण
Summary
इस अध्ययन एक उपन्यास neuronal अक्षतंतु और glia (खगोल) के सह – संस्कृति मंच की स्थापना की प्रक्रिया का वर्णन करता है. इस प्रणाली में सह संस्कृति, एक एकल अक्षतंतु (और एक glial सेल) के बीच सीधी बातचीत के हेरफेर संभव हो जाता है, glial संकेत आपसी न्यूरॉन के यंत्रवत विश्लेषण की अनुमति देता है.
Abstract
Proper neuron to glia interaction is critical to physiological function of the central nervous system (CNS). This bidirectional communication is sophisticatedly mediated by specific signaling pathways between neuron and glia1,2 . Identification and characterization of these signaling pathways is essential to the understanding of how neuron to glia interaction shapes CNS physiology. Previously, neuron and glia mixed cultures have been widely utilized for testing and characterizing signaling pathways between neuron and glia. What we have learned from these preparations and other in vivo tools, however, has suggested that mutual signaling between neuron and glia often occurred in specific compartments within neurons (i.e., axon, dendrite, or soma)3. This makes it important to develop a new culture system that allows separation of neuronal compartments and specifically examines the interaction between glia and neuronal axons/dendrites. In addition, the conventional mixed culture system is not capable of differentiating the soluble factors and direct membrane contact signals between neuron and glia. Furthermore, the large quantity of neurons and glial cells in the conventional co-culture system lacks the resolution necessary to observe the interaction between a single axon and a glial cell.
In this study, we describe a novel axon and glia co-culture system with the use of a microfluidic culture platform (MCP). In this co-culture system, neurons and glial cells are cultured in two separate chambers that are connected through multiple central channels. In this microfluidic culture platform, only neuronal processes (especially axons) can enter the glial side through the central channels. In combination with powerful fluorescent protein labeling, this system allows direct examination of signaling pathways between axonal/dendritic and glial interactions, such as axon-mediated transcriptional regulation in glia, glia-mediated receptor trafficking in neuronal terminals, and glia-mediated axon growth. The narrow diameter of the chamber also significantly prohibits the flow of the neuron-enriched medium into the glial chamber, facilitating probing of the direct membrane-protein interaction between axons/dendrites and glial surfaces.
Protocol
Representative Results
Discussion
MCP आधारित न्यूरॉन और astrocytes सह संस्कृति प्रणाली केवल axons केंद्रीय चैनल के पास अनुमति और astroglial कोशिकाओं के साथ बातचीत के द्वारा अस्थिकणिका संकेत दे रास्ते विस्तृत न्यूरॉन के विच्छेदन की अनुमति देता है. यह सह …
Declarações
The authors have nothing to disclose.
Acknowledgements
हम बीएसी GLT1 EGFP चूहों और GLT1 एंटीबॉडी प्रदान करने के लिए डॉ. Jeffrey रोथस्टीन धन्यवाद देना चाहूंगा, तंत्रिका विज्ञान अनुसंधान के लिए Tufts केंद्र (P30 NIH NS047243, PI, रोब जैक्सन) मूल्यवान कोर की सुविधा प्रदान करने के लिए नए शिक्षकों की भर्ती के अनुदान (P30 5P30NS069254-02 NIH , PI, फिल Haydon Tufts तंत्रिका विज्ञान विभाग में).
Materials
| Fetal bovine serum | Hyclone | SH30070.03 | for plating neuron for neuron cutlure medium |
| Fetal bovine serum | Sigma-Aldrich | F4135 | for astrocyte culture medium |
| Glial derived nerve factor | R&D systems | 212-GD | Apply 10-20 ng/ml to neuron side of chamber |
| Dulbecco modified eagle medium high glucose | Sigma-Aldrich | 11995 | |
| 70 mm cell strainer | BD Falcon | 352350 | |
| Sterile glass bottom dish | MatTek Corporation | ||
| Microfluidic culture platforms | Xona Microfluidics LLC | SND150 | |
| 6 wells of the culture plate | Cellstar | 657 160 | |
Neuron culture medium
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Neuron culture medium for plating cell
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Astrocyte culture medium
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Table 1. Materials used in the microfluidic culture platform-based neuronal axon and glia co-culture system. |
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