Name: tracking hypoxic signaling within encapsulated cell aggregates
Uploaded: 2011-12-16T13:00:00
Duration: 9 min 14 s
Description: Watch this Scientific Journal Video about Tracking Hypoxic Signaling within Encapsulated Cell Aggregates at JoVE.com

उदारता MIN6 कोशिकाओं की आपूर्ति के लिए बोल्डर, कोलोराडो विश्वविद्यालय के Kristi Anseth प्रयोगशाला के लिए धन्यवाद. इस परियोजना के लिए अनुदान NSF द्वारा प्रदान की गई है.

<ol>
	<li>Kelly, W., Lillehei, R., Merkel, F., Idezuki, Y., Goetz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allotransplantation+of+the+pancreas+and+duodenum+along+with+the+kidney+in+diabetic+nephropathy.">Allotransplantation of the pancreas and duodenum along with the kidney in diabetic nephropathy.</a> Surgery. 61, 827-837 (1967).</li><li>Shapiro, J. A. M., Ricordi, C., Hering, B. J., Auchincloss, H., Lindblad, R., Robertson, R. P., Secchi, A., Brendel, M. D., Berney, T., Brennan, D. C., Cagliero, E., Alejandro, R., Ryan, E. A., DiMercurio, B., Morel, P., Polonsky, K. S., Reems, J., Bretzel, R. G., Bertuzzi, F., Froud, T., Kandaswamy, R., Sutherland, D. E. R., Eisenbarth, G., Segal, M., Preiksaitis, J., Korbutt, G. S., Barton, F. B., Viviano, L., Seyfert-Margolis, V., Bluestone, J., Lakey, J. R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+trial+of+the+Edmonton+Protocol+for+islet+transplantation.">International trial of the Edmonton Protocol for islet transplantation.</a> N. Eng. J. Med.. 355, 1318-1330 (2006).</li><li>Sun, A. M., O'Shea, G. M., Goosen, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injectable+microencapsulated+islet+cells+as+a+bioartificial+pancreas.">Injectable microencapsulated islet cells as a bioartificial pancreas.</a> Appli. Biochem. Biotechnol. 10, 87-99 (1984).</li><li>Iwata, H., Amemiva, H., Matsuda, T., Takano, H., Hayashi, R., Akutsu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+microencapsulated+islets+in+agarose+gel+as+bioartificial+pancreas+by+studies+of+hormone+secretion+in+culture+and+by+xenotransplantation.">Evaluation of microencapsulated islets in agarose gel as bioartificial pancreas by studies of hormone secretion in culture and by xenotransplantation.</a> Diabetes. 39, 224-225 (1989).</li><li>Zhang, W. J., Laue, C., Hyder, A., Schrezenmeir, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purity+of+alginate+affects+the+viability+and+fibrotic+overgrowth+of+encapsulated+porcine+iselt+xenografts.">Purity of alginate affects the viability and fibrotic overgrowth of encapsulated porcine iselt xenografts.</a> Transplant Proc. 33, 3517-3519 (2001).</li><li>Lin-Gibson, S., Bencherif, S., Cooper, J. A., Wetzel, S. J., Antonucci, J. M., Vogel, B. M., Horkay, F., Washburn, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+characterization+of+PEG+dimethacrylates+and+their+hydrogels.">Synthesis and characterization of PEG dimethacrylates and their hydrogels.</a> Biomacromolecules. 5, 1280-1287 (2004).</li><li>Weber, L. M., He, J., Bradley, B., Haskins, K., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PEG-based+hydrogels+as+an+in+vitro+encapsulation+platform+for+testing+controlled+&#x03B2;-cell+microenvironments.">PEG-based hydrogels as an in vitro encapsulation platform for testing controlled &#x03B2;-cell microenvironments.</a> Acta. Biomater. 2, 1-8 (2006).</li><li>Weber, L. M., Lopez, C. G., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+PEG+hydrogel+crosslinking+density+on+protein+diffusion+and+encapsulated+islet+survival+and+function.">Effects of PEG hydrogel crosslinking density on protein diffusion and encapsulated islet survival and function.</a> J. Biomed. Mater. Res. A. 90, 720-729 (2009).</li><li>Bryant, S. J., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+properties+influence+ECM+production+by+chondrocyte+photoencapsulated+in+poly(ethylene+glycol)+hydrogels.">Hydrogel properties influence ECM production by chondrocyte photoencapsulated in poly(ethylene glycol) hydrogels.</a> J. Biomed. Mater. Res. 59, 63-72 (2001).</li><li>Bryant, S. J., Nuttelman, C. R., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytocompatibility+of+UV+and+visible+light+photoinitiating+systems+on+cultured+NIH/3T3+fibroblasts+in+vitro.">Cytocompatibility of UV and visible light photoinitiating systems on cultured NIH/3T3 fibroblasts in vitro.</a> J. Biomater. Sci. Polymer Edn. 11, 439-457 (2000).</li><li>Dionne, K. E., Colton, C. K., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+oxygen+on+isolated+pancreatic+tissue.">Effect of oxygen on isolated pancreatic tissue.</a> Trans. Am. Soc. Artif. Intern. Organs. 35, 739-741 (1989).</li><li>Dionne, K. E., Colton, C. K., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+hypoxia+on+insulin+secretion+by+isolated+rat+and+canine+islets+of+Langerhans.">Effect of hypoxia on insulin secretion by isolated rat and canine islets of Langerhans.</a> Diabetes. 42, 12-21 (1993).</li><li>De Groot, M., Schuurs, T. A., Keizer, P. P. M., Fekken, S., Leuvenink, H. G. D., van Schilfgaarde, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+encapsulated+rat+pancreatic+islets+to+hypoxia.">Response of encapsulated rat pancreatic islets to hypoxia.</a> Cell Transplant. 12, 867-875 (2003).</li><li>Fancy, M. L., Topiwala, R., Sahai, P., S, J. O., Blanchette, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlating+hypoxia+with+insulin+secretion+using+a+fluorescent+hypoxia+detection+system.">Correlating hypoxia with insulin secretion using a fluorescent hypoxia detection system.</a> J. Biomed. Mater. Res. B. 97, 148-155 (2011).</li><li>Webb, J. D., Coleman, M. L., Pugh, C. W. H. y. p. o. x. i. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hypoxia-inducible+factors+(HIF),+HIF+hydroxylases+and+oxygen+sensing.">hypoxia-inducible factors (HIF), HIF hydroxylases and oxygen sensing.</a> Cell. Mol. Life Sci. 66, 3539-3554 (2009).</li></ol>

ब्याज की कोई संघर्ष की घोषणा की.

यहाँ प्रस्तुत विधि गैर शारीरिक स्थितियों के न्यूनतम उपयोग के साथ एक खूंटी hydrogel में सेल encapsulation के लिए एक त्वरित और सरल तकनीक प्रदान करता है. खूंटी और biocompatibility संशोधन की आसानी के लिए एक बहुत ही उपयोगी सामग्री encapsulation का प्रतिनिधित्व करता है. प्रकाश द्वारा सहज प्रभावित समाधान में खूंटी प्रतिशत के साधारण परिवर्तन, उदाहरण के लिए, रोमकूप आकार के माध्यम से compressive मापांक के रूप में यांत्रिक गुणों, और परिवहन संपत्तियों को समायोजित करने के लिए इस्तेमाल किया हो सकता है. इसके अलावा, खूंटी पक्ष श्रृंखला के अलावा द्वारा आसानी से संशोधित किया गया है. खूंटी hydrogels, इसलिए दोनों एक आशाजनक नैदानिक ​​डिवाइस और इन विट्रो अनुसंधान में के लिए एक लचीला मंच का प्रतिनिधित्व खूंटी समझाया कोशिकाओं में hypoxia पर नज़र रखने के लिए एक विधि को भी प्रस्तुत किया गया. इस विधि hypoxia का पता लगाने के सादगी के लिए और ब्याज की कोशिकाओं को बलिदान की जरूरत से बचने के लिए उपयोगी है. तकनीक कोशिकाओं के प्रकार की एक किस्म के लिए हमें अपनी स्थितियों की एक किस्म में लागू किया जा सकता हैव्यापक efulness. उदाहरण के लिए, स्टेम सेल भेदभाव के लिए एक क्यू के रूप में hypoxia स्टेम सेल micromass संस्कृतियों में लगाया जा सकता है. हालांकि, इस विधि केवल सेल सिस्टम या सिस्टम में जो कोशिकाओं छितरी बाद में एकत्रित कर रहे हैं को फैलाने के लिए लागू किया जा सकता है. इसके अलावा, फ्लोरोसेंट संकेत का पता लगाने बड़ा या denser ऊतकों में मुश्किल हो सकता है.

<table border="1"><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> सूचीपत्र संख्या </td><td> टिप्पणियां (वैकल्पिक </td></tr><tr><td> खूंटी </td><td> सिग्मा Aldrich </td><td> 309,028-500G </td><td></td></tr><tr><td> Methacrylic Anhydride </td><td> सिग्मा Aldrich </td><td> 276,685-100ml </td><td></td></tr><tr><td> माइक्रोवेव </td><td> एमर्सन </td><td> MW8784SB </td><td></td></tr><tr><td> Vortexer </td><td> वैज्ञानिक इंडस्ट्रीज </td><td> एसआई-A236 </td><td></td></tr><tr><td> Methylene क्लोराइड </td><td> सिग्मा Aldrich </td><td> D65100-1L </td><td></td></tr><tr><td> Diethyl ऐतेर </td><td> सिग्मा Aldrich </td><td> 346,136-1L </td><td></td></tr><tr><td> डायलिसिस टयूबिंग </td><td> स्पेक्ट्रम </td><td> 132640 </td><td></td></tr><tr><td></td><td> प्रयोगशालाओं </td><td></td><td></td></tr><tr><td> फ्रीजर </td><td></td><td></td><td></td></tr><tr><td&gt; Lyophilizer </td><td> Labconco </td><td> 7670521 </td><td></td></tr><tr><td> वेक्यूम - पंप </td><td> वेल्श </td><td> 8917Z-01 </td><td></td></tr><tr><td> 2959 Irgacure </td><td> Ciba - Geigy </td><td> 029891301PS04 </td><td></td></tr><tr><td> HBSS </td><td> Mediatech </td><td> 21-022-CV </td><td></td></tr><tr><td> सिरिंज फ़िल्टर </td><td> VWR </td><td> 28145-477 </td><td></td></tr><tr><td> 1640 RPMI </td><td> Mediatech </td><td> * </td><td> * कस्टम तैयार </td></tr><tr><td> FBS </td><td> Paa प्रयोगशालाओं </td><td> A15-351 </td><td></td></tr><tr><td> पेनिसिलिन - स्ट्रेप्टोमाइसिन </td><td> Mediatech </td><td> 30 002-CI </td><td></td></tr><tr><td> Amphotericin बी </td><td> Mediatech </td><td> 30-003-सीएफ़ </td><td></td></tr><tr><td> इनक्यूबेटर </td><td> थर्मो वैज्ञानिक </td><td> 3597 </td><td> NAPCO श्रृंखला 8000 WJ दमन w / O2 </td></tr><tr><td> Trypsin EDTA </td><td> Mediatech </td><td> 25 052-CI </td><td></td></tr><tr><td> कक्षीय शेखर</td><td> VWR </td><td> 12620-926 </td><td></td></tr><tr><td> यूवी लैम्प </td><td> Sanyo Denri </td><td> / FLR40SBLB एम </td><td> 40W दो, 365nm blacklight नीले यूवी बल्ब रखती है </td></tr><tr><td> अपकेंद्रित्र </td><td> Eppendorf </td><td> 5811 000.010 * </td><td> * क्रम संख्या. मॉडल 5810 आर </td></tr><tr><td> खुर्दबीन </td><td> Nikon </td><td> तिवारी-ND6-PFS </td><td> 556nm उत्तेजना / 586nm उत्सर्जन के लिए filterset के साथ </td></tr></table>

tracking hypoxic signaling within encapsulated cell aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

Watch this Scientific Journal Video about Tracking Hypoxic Signaling within Encapsulated Cell Aggregates at JoVE.com

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

Crosslinked खूंटी hydrogel में कक्षों की तस्वीर encapsulation के लिए एक विधि का वर्णन है. समझाया murine insulinoma भीतर hypoxic संकेतन (MIN6) समुच्चय एक फ्लोरोसेंट मार्कर प्रणाली का उपयोग करने के लिए नज़र रखी है. इस प्रणाली के एक hydrogel पाड़ और hypoxic संकेत के सहसंबंध के भीतर सेल phenotype में परिवर्तन के साथ कोशिकाओं के धारावाहिक परीक्षा की अनुमति देता है.

एनकेप्सुलेटेड सेल समुच्चय के भीतर ट्रैकिंग hypoxic संकेतन

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

Research

JoVE Journal

Bioengineering

3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

एक संशोधित दो photon माइक्रोस्कोप में 3 डी कक्षीय ट्रैकिंग: Intracellular vesicles की ट्रैकिंग के लिए एक आवेदन

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

एक तीन आयामी मैट्रिक्स कोलेजन के भीतर सेल प्रवास का विश्लेषण

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

के लिए एक उपकरण के रूप में एक लिपोसोमल-समझाया पास अवरक्त की fluorophore प्रतिदीप्ति शमन<em&gt; Vivo</em&gt; ऑप्टिकल इमेजिंग

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

प्रतिदीप्ति Biomembrane फोर्स जांच: एक एकल कोशिका पर रिसेप्टर ligand के काइनेटिक्स की समवर्ती Quantitation और बाध्यकारी प्रेरित Intracellular संकेतन

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions ...

Transport Properties of Ibuprofen Encapsulated in Cyclodextrin Nanosponge Hydrogels: A Proton HR-MAS NMR Spectroscopy Study

Cyclodextrin Nanosponge hydrogels में Ibuprofen समझाया की परिवहन गुण: एक प्रोटॉन एचआर-मास एनएमआर स्पेक्ट्रोस्कोपी अध्ययन

The chemical cross-linking of &#946;-cyclodextrin (&#946;-CD) with ethylenediaminetetraacetic dianhydride (EDTA) led to branched polymers referred to as ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

इंजीनियरिंग तीन आयामी उपकला ऊतकों कोशिकी मैट्रिक्स के भीतर एम्बेडेड

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

एक लिक्विड क्रिस्टल Nanoparticle कैरियर में एक हाइड्रोफोबिक कार्गो समझाया की लक्षित प्लाज्मा झिल्ली प्रसव

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

एक Microwell Array प्लेटफॉर्म के भीतर विधानसभा और माइक्रोबियल सामुदायिक विकास का ट्रैकिंग

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

तीन आयामी के भीतर निस्र्पक कक्ष माइग्रेशन इन विट्रो घाव वातावरण

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

A Protocol for Real-time 3D Single Particle Tracking

रीयल-टाइम 3d एकल कण ट्रैकिंग के लिए एक प्रोटोकॉल

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...