Name: tunable hydrogels from pulmonary extracellular matrix for 3d cell culture
Uploaded: 2017-01-17T13:00:00
Description: Watch this Scientific Journal Video about Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture at JoVE.com

Nous tenons à remercier les fermes de Smithfield pour le don du tissu pulmonaire porcin intact. Nous tenons également à remercier le Dr Hu Yang, le Dr Christina Tang et le département de chirurgie plastique VCU pour nous permettre d&#39;utiliser leur équipement. échantillons hydrogels et de tissus ont été préparés pour SEM au Département VCU d&#39;anatomie et de l&#39;installation de neurobiologie Microscopie pris en charge, en partie, grâce au financement du NIH-NINDS Core Center Grant 5 P30 NS047463 et, en partie, par la forme de financement du NIH-NCI Cancer Support Center Grant P30 CA016059. SEM imagerie d&#39;échantillons à l&#39;installation de base Caractérisation VCU Nanotechnology (CCN). Ce travail a été financé par la National Science Foundation, CMMI 1351162.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Solution </td><td> Filtre stérile </td><td> instructions </td></tr><tr><td> Dih 2 O </td><td> Oui </td><td> Dih 2 O; filtré de manière stérile </td></tr><tr><td> Solution à 0,1% de Triton X-100 </td><td> Oui </td><td> Sous la hotte ajouter 100 pi de Triton-X 100 Solution 100 ml Dih 2 O et agiter jusqu&#39;à dissolution; filtre stérile. </td></t...

<ol>
	<li>Lee, J., Cuddihy, M., Kotov, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Three-Dimensional Cell Culture Matrices: State of the Art. 14, (2008).</li><li>Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 3D Cell Culture. , (2011).</li><li>Walker, J. M., Ed, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Epithelial cell culture protocols. , (2012).</li><li>Badylak, S. F., Freytes, D. O., Gilbert, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+as+a+biological+scaffold+material:+Structure+and+function.">Extracellular matrix as a biological scaffold material: Structure and function.</a> Acta Biomater. 5 (1), 1-13 (2009).</li><li>Koo, H. -. J., Lim, K. -. H., Jung, H. -. J., Park, E. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+evaluation+of+gardenia+extract,+geniposide+and+genipin.">Anti-inflammatory evaluation of gardenia extract, geniposide and genipin.</a> J. Ethnopharmacol. 103 (3), 496-500 (2006).</li><li>Jeffords, M. E., Wu, J., Shah, M., Hong, Y., Zhang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+Material+Properties+of+Cardiac+Matrix+Hydrogels+to+Induce+Endothelial+Differentiation+of+Human+Mesenchymal+Stem+Cells.">Tailoring Material Properties of Cardiac Matrix Hydrogels to Induce Endothelial Differentiation of Human Mesenchymal Stem Cells.</a> ACS Appl. Mater. Interfaces. 7 (20), 11053-11061 (2015).</li><li>Suckow, M. A., Voytik-Harbin, S. L., Terril, L. A., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+bone+regeneration+using+porcine+small+intestinal+submucosa.">Enhanced bone regeneration using porcine small intestinal submucosa.</a> J. Invest. Surg. 12 (5), 277-287 (1998).</li><li>Brown, B. N., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+as+an+inductive+scaffold+for+functional+tissue+reconstruction.">Extracellular matrix as an inductive scaffold for functional tissue reconstruction.</a> Transl. Res. J. Lab. Clin. Med. 163 (4), 268-285 (2014).</li><li>Pouliot, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+a+naturally+derived+lung+extracellular+matrix+hydrogel.">Development and characterization of a naturally derived lung extracellular matrix hydrogel.</a> J.Biomed.Mater.Res.A. , (2016).</li><li>Price, A. P., England, K. A., Matson, A. M., Blazar, B. R., Panoskaltsis-Mortari, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+decellularized+lung+bioreactor+system+for+bioengineering+the+lung:+the+matrix+reloaded.">Development of a decellularized lung bioreactor system for bioengineering the lung: the matrix reloaded.</a> Tissue Eng. Part A. 16 (8), 2581-2591 (2010).</li><li>Freytes, D. O., Martin, J., Velankar, S. S., Lee, A. S., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+rheological+characterization+of+a+gel+form+of+the+porcine+urinary+bladder+matrix.">Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix.</a> Biomaterials. 29 (11), 1630-1637 (2008).</li><li>Tilghman, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+rigidity+regulates+cancer+cell+growth+and+cellular+phenotype.">Matrix rigidity regulates cancer cell growth and cellular phenotype.</a> PloS One. 5 (9), e12905 (2010).</li><li>Ratner, B. D., Bryant, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials:+where+we+have+been+and+where+we+are+going.">Biomaterials: where we have been and where we are going.</a> Annu. Rev. Biomed. Eng. 6, 41-75 (2004).</li><li>Fridman, R., Benton, G., Aranoutova, I., Kleinman, H. K., Bonfil, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+initiation+and+growth+of+tumor+cell+lines,+cancer+stem+cells+and+biopsy+material+in+mice+using+basement+membrane+matrix+protein+(Cultrex+or+Matrigel)+co-injection.">Increased initiation and growth of tumor cell lines, cancer stem cells and biopsy material in mice using basement membrane matrix protein (Cultrex or Matrigel) co-injection.</a> Nat Protoc. 7 (6), 1138-1144 (2012).</li><li>Zhang, W. -. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reconstruction+of+lung+alveolus-like+structure+in+collagen-matrigel/microcapsules+scaffolds+in+vitro.">The reconstruction of lung alveolus-like structure in collagen-matrigel/microcapsules scaffolds in vitro.</a> J. Cell. Mol. Med. 15 (9), 1878-1886 (2011).</li><li>Booth, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acellular+Normal+and+Fibrotic+Human+Lung+Matrices+as+a+Culture+System+for+In+Vitro+Investigation.">Acellular Normal and Fibrotic Human Lung Matrices as a Culture System for In Vitro Investigation.</a> Am. J. Respir. Crit. Care Med. 186 (9), 866-876 (2016).</li><li>Matsuda, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+human+lung+stem+cells.">Evidence for human lung stem cells.</a> N. Engl. J. Med. 364 (19), 1795-1806 (2011).</li><li>Zuo, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p63+Krt5++distal+airway+stem+cells+are+essential+for+lung+regeneration.">p63+Krt5+ distal airway stem cells are essential for lung regeneration.</a> Nature. 517 (7536), 616-620 (2015).</li><li>Keane, T. J., Londono, R., Turner, N. J., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+ineffective+decellularization+of+biologic+scaffolds+on+the+host+response.">Consequences of ineffective decellularization of biologic scaffolds on the host response.</a> Biomaterials. 33 (6), 1771-1781 (2012).</li><li>Neill, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+of+human+and+porcine+lung+tissues+for+pulmonary+tissue+engineering.">Decellularization of human and porcine lung tissues for pulmonary tissue engineering.</a> Ann Thorac Surg. 96 (3), 1046-1055 (2013).</li></ol>

Un des aspects les intégrales de la biologie est l&#39;auto-organisation des molécules dans les structures hiérarchiques qui effectuent une tâche spécifique. 13 En laboratoire, l&#39; auto-assemblage dépend de nombreux facteurs tels que la concentration en sel, le pH et la durée de digestion. Comme le montre, il se forme d&#39;hydrogel auto-organisation lorsque les protéines solubilisées retour à une température physiologique. L&#39;hydrogel formé est capable de favoriser l&#39; attachement et la ...

Translating in vitro results to the clinic is one of the most challenging issues facing biomedical researchers. In vitro research on tissue culture plastic is easier, more convenient, and maintains high cell viability.1 This approach is a reasonable starting point, but the results have limited clinical translation. Increasingly, laboratories are incorporating three-dimensional constructs to replace the traditional two-dimensional methods. Reviews are available for many three-dimensional environments, from biological scaffolds to polymeric scaffolds.2,3
Biological frameworks can mimic characteristics of in vivo environments as they contain many of the protein and glycosaminoglycan components of the native matrix and provide familiar binding sites for cells to attach to and recognize. Extracellular matrix (ECM) derived materials have been shown to be capable scaffolds for cell attachment and proliferation.4 One challenge that limits the application of ECM hydrogel platforms stems from their inherently weak mechanical properties following gelation. Native tissue often has mechanical properties that are magnitudes higher than hydrogels. Non-toxic crosslinking agents can increase the mechanical properties of hydrogels to better mimic the native tissue environment. Genipin is a non-toxic, natural crosslinker derived from Gardenia plants with the ability to closely tailor mechanical properties of ECM with changes in genipin concentration5,6. 
Nearly all cells in the body exist in, and organize on, ECM that they either produce or maintain. New focus on the universal importance of ECM in the organization, condition, and function in every organ or system has sparked the production of matrix based platforms for in vitro investigation. Porcine small intestine submucosa is the most extensively studied naturally-derived scaffold, and it has been used to regenerate tendons, ligaments, skeletal muscle4, and even bone7. Matrices from other organs and donor species have also demonstrated good tissue regeneration potential. The use of foreign ECM components causes minimal issues with immunomodulation. After elimination of host cellular matter, the remaining ECM will be similar in amino acid content and organization to all other mammalian species8. There is a growing line of thinking that the best way to examine cell-ECM interactions in vitro is to utilize organ-specific ECM scaffolds. Each organ provides a unique composition of proteins and proteoglycans to create cellular niches. Niches provide structural, functional and even the enzymatic breakdown of the extracellular matrix contributing to biophysical signaling. To attain an in vitro microenvironment most similar to the in vivo microenvironment, use of tissue specific ECM would optimize the cellular niches for research. 
The goal of this protocol is to provide a method for establishing a hydrogel scaffold unique to the lung ECM. This method provides a platform for in vitro research on lung cell-ECM interactions.

<table border="0" cellpadding="0" cellspacing="0" width="841">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton X-100&#160;</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">BP151-100</td>
			<td style="width:371px;">Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Deoxycholate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">D6750-100g</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7506-500g</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1016-500g</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>D5025-150KU</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCl</td>
			<td>Sigma-Aldrich</td>
			<td>258148-500ML</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pepsin</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td>P6887-5G</td>
			<td>Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Hydroxide</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">BP359-500</td>
			<td>Use with eye protection and gloves.</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Genipin</td>
			<td style="width:135px;">Wako Chemicals</td>
			<td style="width:119px;">078-03021</td>
			<td>Use in fume hood with eye protection and gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS 10x</td>
			<td style="width:135px;">Quality Biological</td>
			<td>119-069-151</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td style="width:135px;">VWR</td>
			<td>45000-448</td>
			<td>None</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter Paper</td>
			<td>Whatman</td>
			<td align="right">8519</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hand pump</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td>10-239-1</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Graduate Beaker</td>
			<td style="width:135px;">VitLab</td>
			<td align="right" style="width:119px;">445941</td>
			<td>N/A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryomill</td>
			<td>SPEX</td>
			<td>6700</td>
			<td>Use cryogloves and eye protection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lyophilizer</td>
			<td>FTS FlexiDry</td>
			<td></td>
			<td>Use gloves.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rheometer</td>
			<td>Discovery</td>
			<td>HR-2</td>
			<td>Use gloves and eye protection.</td>
		</tr>
	</tbody>
</table>

tunable hydrogels from pulmonary extracellular matrix for 3d cell culture

Nous présentons ici une méthode pour établir des composants multiples hydrogels de culture cellulaire pour la culture in vitro de cellules de poumon. En commençant par la santé en tissu bloc pulmonaire de porc, rat ou la souris, le tissu est perfusé et immergé dans les détergents chimiques ultérieures pour éliminer les débris cellulaires. comparaison histologique du tissu avant et après le traitement confirme l&#39;élimination de plus de 95% de la coloration de l&#39;ADN double brin et l&#39;alpha-galactosidase suggère que la majorité des débris cellulaires sont éliminés. Après décellularisation, le tissu est lyophilisé, puis cryomilled en poudre. La poudre de matrice est digéré pendant 48 heures dans une solution de digestion de la pepsine acide, puis on neutralise pour former la solution de pré-gel. Gélification de la solution de pré-gel peut être induite par une incubation à 37 ° C et peut être utilisée immédiatement après la neutralisation ou stocké à 4 ° C pendant jusqu&#39;à deux semaines. Les revêtements peuvent être formés en utilisant la solution de pré-gel sur une plaque non-traitée pour cattachement ell. Les cellules peuvent être mises en suspension dans le pré-gel avant de l&#39;autoassemblage de réaliser une culture en 3D, plaquée sur la surface d&#39;un gel formé à partir de laquelle les cellules peuvent migrer à travers l&#39;échafaudage, ou plaquée sur les revêtements. Les modifications de la stratégie présentés peuvent avoir un impact température de gélification, la force ou la taille des fragments de protéines. Au-delà de la formation d&#39;hydrogel, la rigidité d&#39;hydrogel peut être augmentée en utilisant genipin.

En utilisant cette méthode, nous avons produit des hydrogels de porc normale, rat, et les poumons de souris (Figure 1). poumons transformés fournissent environ 5 mg, 40 mg et 10 g de poudre ECM respectivement. Un aperçu du procédé est représenté sur la figure 2. visualisations clé au cours du processus comprennent: aspect blanc des poumons après le rinçage désoxycholate; après la formation de pré-gel, une solution doit être opaque et la sol...

Watch this Scientific Journal Video about Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture at JoVE.com

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Ceci est une méthode pour créer un échafaudage trois dimensions de culture de cellules de la matrice extracellulaire pulmonaire. poumon Intact est transformé en hydrogels qui peuvent soutenir la croissance des cellules en trois dimensions.

Accordable hydrogels de matrice extracellulaire pulmonaire pour la 3D Culture Cellulaire

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Research

JoVE Journal

Bioengineering

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Formation et Patterning Light-médiée hydrogels pour la culture cellulaire Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Recherche

Bio-ingénierie

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Fabrication de Mousses et microsupports dérivés Matrice extracellulaires culture cellulaire spécifique de tissu et plates-formes de livraison

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Fabrication d’un Hydrogel de dérivés de matrice extracellulaire rein Cortex

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Production des fibres de la matrice extracellulaire par Culture de cellules de Membrane fibre creuse sacrificiel

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Mesure des métalloprotéinases de la matrice cellulaire Global et l’activité métabolique des Hydrogels 3D

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

Bioink extracellulaire de matrice de tissu pancréatique-dérivé pour l'impression 3D Cell-Laden Pancréatique Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

Un modèle de culture matrix-adipocyte extracellulaire humaine 3D pour étudier matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Préparation des microenvironnements de matrice extracellulaire tunable pour évaluer les spécifications du phénotype de cellules de Schwann

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Microgel-Extracellular Matrix Composite Support for the Embedded 3D Printing of Human Neural Constructs

Support composite à matrice microgel-extracellulaire pour l’impression 3D intégrée de constructions neuronales humaines

The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication ...

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels (Video) | JoVE

Bio-impression 3D d’hydrogels photoaccordables pour étudier l’activation des fibroblastes

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...