Vorremmo ringraziare le aziende agricole Smithfield per la donazione di tessuto polmonare suino intatto. Vorremmo anche ringraziare il Dr. Hu Yang, il Dr. Christina Tang e Chirurgia Dipartimento di VCU plastica per averci permesso di utilizzare le loro attrezzature. campioni di idrogel e di tessuto sono stati preparati per SEM presso il Dipartimento VCU di Anatomia e Neurobiologia Microscopia strumento sostenuto, in parte, da un finanziamento NIH-NINDS nucleo centrale di Grant 5 P30 NS047463 e, in parte, dalla forma di finanziamento NIH-NCI Cancer Support Center di Grant P30 CA016059. l&#39;imaging SEM dei campioni presso il VCU Nanotecnologia Nucleo Caratterizzazione Facility (NCC). Questo lavoro è stato finanziato dalla National Science Foundation, CMMI 1.351.162.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Soluzione </td><td> filtro sterile </td><td> Indicazioni </td></tr><tr><td> DIH 2 O </td><td> sì </td><td> DIH 2 O; sterile filtrato </td></tr><tr><td> 0,1% soluzione di Triton X-100 </td><td> sì </td><td> Sotto cappa aggiungere 100 ml Triton-X 100 Soluzione a 100 ml DIH 2 O e agitare fino alla dissoluzione; filtro sterile. </td></tr><tr><td> 2% Desossicolato Soluzione </td><td> sì </td><td> Sotto cappa aggiungere 2 g di sodio soluzione Desossicolato per 100 ml DIH 2 O e agitare fino alla dissoluzione; filtro sterile </td></tr><tr><td> 1 M NaCl </td><td> sì </td><td> Aggiungere 58,44 g di NaCl a 1 L di DIH 2 O; agitare fino a scioglimento; filtro sterile </td></tr><tr><td> soluzione DNasi </td><td> sì </td><td> Aggiungere 12.000unità di DNasi a 1 L DIH 2 O; Aggiungere 0,156 g MgSO 4 (Anidro), e 0,222 g CaCl 2; agitare fino a scioglimento; filtro sterile </td></tr><tr><td> PBS </td><td> sì </td><td> Unire 27,2 g di Na 2 HPO 4 · 7H 2 O (bibasico eptaidrato), 80 g di NaCl, e 2 g KCl con 10 L DIH 2 O; agitare fino a scioglimento; aggiustare il pH a 7,4; filtro sterile </td></tr></tbody></table> Tabella 1: soluzioni necessarie per Tissue Decellularization. Effettuare le soluzioni di cui sopra per il processo di decellularization. Conservare a 4 ° C. Circa 2,5 L sarà necessario per un polmone suino. 1. Formazione Hydrogel <ol><li> Suina Lung Decellularization (adattato da riferimenti 9,10) <ol><li> Ottenere un blocco normale polmone suino, da un macello, con il cuore e Vasculature intatto. </li><li> Utilizzare forbici di tessuto o un bisturi per sezionare il cuore, vasi più vicino possibile al cuore di taglio, la vascolarizzazione sarà utilizzato in fasi successive per perfusione. </li><li> sezionare cura di collocare il tessuto connettivo circostante la trachea, bronchi e vasi con un bisturi o forbici. NOTA: determinare le migliori polmone da utilizzare per il resto della procedura con la scelta di un polmone, senza grandi tagli o punture attraverso la pleura e grandi quantità di atelettasia o occlusione vascolare che possono ostacolare l&#39;efficacia del processo di decellularization. </li><li> Tagliare il polmone non ottimale lasciando tanto bronchi attaccati alla trachea possibile (cuore, tessuto connettivo e i lobi del polmone rimosso può essere smaltito). Ci dovrebbe essere un polmone rimanendo attaccata alla trachea e vascolare. NOTA: Conservare sezioni per istologia se lo si desidera. 9 </li><li> Chiudere bronchi scollegati con pinze o sutura per prevent riflusso eccesso. </li></ol><ol start="6"><li> Preparare le soluzioni decellularization e conservare in frigorifero a 4 ° C fino al momento (Tabella 1). </li><li> Utilizzando una pompa a mano cannulata per adattare l&#39;arteria polmonare, profumato il tessuto polmonare 3 volte con acqua deionizzata sia attraverso l&#39;arteria polmonare e la trachea. Profumato vascolarizzazione prima volta in volta. Iniziare con circa 1 L nel sistema vascolare e 1,5 L in trachea per ogni perfusione, ma come detriti cellulari viene rimosso più liquido può essere perfusi attraverso il sistema. </li><li> Profumato sia vascolarizzazione e la trachea con Triton X-100 soluzione. </li><li> Immergere tessuto polmonare in Triton X-100 soluzione per 24 ore a 4 ° C. </li><li> Perfuse vascolare e trachea 3 volte con acqua deionizzata per sciacquare. </li><li> Profumato sia vascolarizzazione e la trachea con la soluzione desossicolato. </li><li> Immergere le sezioni di tessuto in desossicolato per 24 ore a 4 ° C. </li><li> Perfuse vascolare e trachea 3 volte con acqua deionizzata per sciacquare. </li><li>Profumato sia vascolarizzazione e la trachea con soluzione di NaCl. </li><li> Immergere il tessuto in soluzione NaCl filtrata per 1 ora a 4 ° C. </li><li> Perfuse vascolare e trachea 3 volte con acqua deionizzata per sciacquare. </li><li> Profumato sia vascolarizzazione e la trachea con una soluzione di DNasi. </li><li> Immergere il tessuto in soluzione DNasi filtrata per 1 ora a 4 ° C. </li><li> Profumato sia vascolare e la trachea 5 volte con PBS. </li><li> Sezionare via tessuto cartilagineo evidente, trachea e tutto tubuli 2 mm o più di diametro (che si trova principalmente attorno l&#39;ilo e porzioni mediali dei polmoni) di condurre le vie aeree, lasciando solo le zone respiratorie (soprattutto le aree periferiche). </li><li> Sezionare il tessuto in 1 sezioni pollici o più piccoli. Orientamento del tessuto non ha importanza per questo passo. </li><li> Eliminare il tessuto liquido in eccesso e in 50 ml provette coniche. Congelare il tessuto a -80 ° C. NOTA: Mantieni sezioni per l&#39;esame istologico per garantire la rimozione di cellule e debri cellulares, se desiderato. Usiamo H &amp; E, α-galattosidasi, PicoGreen, idrossiprolina, ninidrina, Alcian blu, SDS-PAGE, e spettrometria di massa per la caratterizzazione. 9 </li></ol></li><li> Lung Processing <ol><li> Rimuovere i coperchi da tubi congelati contenenti tessuto polmonare decellularized. </li><li> Mettere carta da filtro sopra l&#39;apertura del tubo e fissare con nastro di gomma. NOTA: Tube e contenuto deve ancora essere congelati comunque di trovarsi in -80 ° C fino congelati. </li><li> Lyophilize il tessuto fino a quando tutto il liquido in eccesso è andato, utilizzando un liofilizzatore secondo le istruzioni del produttore. Conservare a -80 ° C fino al momento di mulino. </li><li> Prima di iniziare aggiungere azoto liquido fresare freezer per raffreddare isolamento e componenti interni alle condizioni di lavoro. </li><li> Rimuovere la barra mulino magnetica dal tubo di mulino e aggiungere il tessuto per coprire fondo. </li><li> Sostituire bar mulino e aggiungere il tessuto poco compresso. La barra mulino deve ancora muoversi liberamente. </li><li> Chiudere la provetta mulino e PLAce nel mulino congelatore. </li><li> Riempire azoto liquido per la linea di riempimento massimo. </li><li> Mulino Congelatore tutto il tessuto in polvere fine (circa 5 minuti, il tasso di compressione pari a ~ 600 min -1), in un cilindro di policarbonato con un dispositivo di simulazione in acciaio inox così come tappi terminali in acciaio inox con il mulino congelatore secondo le istruzioni del produttore. Conservare a -80 ° C fino al momento dell&#39;uso. </li></ol></li><li> Micro-porosa formazione di gel (8 mg / ml) (adattato da riferimenti 9,11) <ol><li> Aggiungere 1% (w / v) di polvere decellularized e 0,1% (w / v) di pepsina per 0,01 M HCl, sotto costante agitazione (dovrebbe essere in grado di vedere il flusso a livello superiore del liquido), a temperatura ambiente, per 48 hr. NOTA: La polvere viene caricata staticamente, in modo da aggiungere il HCl dopo la polvere offre la possibilità di lavare l&#39;eccesso al largo delle pareti del tubo. </li><li> Dopo la digestione per 48 h, porre la soluzione e reagenti in ghiaccio per 5 min. </li><li> Utilizzando refrigerato 10% (v / v) 0,1 M NaOH, und 11.11% (v / v) 10x PBS (per portare l&#39;intera soluzione a 1x concentrazione), portare la soluzione proteica digerita a pH fisiologico di 7,4. NOTA: La soluzione può essere conservato a 4 ° C per una settimana. Eseguire la cinetica di gelificazione utilizzando reometria 9 se lo si desidera. </li></ol></li></ol> 2. Cross-collega idrogeli per migliorare la resistenza meccanica <ol><li> Soluzioni di 1%, 0,1% e 0,01% w / v soluzione genipina reticolazione sono state preparate sciogliendo la necessaria quantità di polvere genipina in 10% DMSO. </li><li> Agitare la soluzione ogni 15 min per 1 ora. </li><li> Aggiungere la soluzione genipina per coprire idrogel ECM (100 microlitri per ciascun pozzetto di una piastra a 96 pozzetti) che sono stati precedentemente assemblati in parte 1.3.4. </li><li> Lasciare ogni idrogel ECM per reticolare per 24 ore a 37 ° C. </li><li> Risciacquare l&#39;idrogel ECM 3 volte con PBS fino a quando la soluzione di lavaggio non è più blu. Gli idrogel reticolati saranno quindi pronto per l&#39;ulteriore caratterizzazione e cestudi su colture ll. </li></ol> 3. coltura cellulare con microporosa Gel <ol><li> Rivestimento bidimensionale <ol><li> Utilizzando la soluzione dalla 1.3.3, aggiungere 20 ml di soluzione pregel a ciascun pozzetto di una piastra di coltura tissutale a 96 pozzetti non trattate. </li><li> durante la notte in frigorifero a 4 ° C per consentire la proteina adsorbimento al piatto. </li><li> Aspirare soluzione PreGel e risciacquare con PBS. </li><li> Cellule Passage 9. </li><li> Aggiungere 10.000 cellule / cm 2 per pozzi. </li><li> Aumentare la media per 100 pl per pozzetto e incubare a 37 ° C. </li></ol></li><li> Tre coltura cellulare dimensionale 9 <ol><li> Utilizzando la soluzione da 1.3.3, risospendere le cellule pellet ad una concentrazione di 1.000.000 cellule / ml di soluzione pregel. </li><li> erogare rapidamente 16 ml di sospensione PreGel cellule in ciascun pozzetto di una piastra a 96 pozzetti, in modo da formare un idrogel di spessore ~ ​​500 micron per la coltura delle cellule 3D. </li><li> Incubare a 37 ° C per 30min per il gel per formare. </li><li> Aggiungere 100 microlitri di media all&#39;inizio idrogel formati e incubare a 37 ° C. </li></ol></li><li> Tre coltura cellulare tridimensionale su gel di rigidità variabili <ol><li> Utilizzando la soluzione da 1.3.3, pipetta 100 ml di soluzione di PreGel in ciascun pozzetto di una piastra da 96 pozzetti. </li><li> Incubare a 37 ° C per 30 minuti per gel di forma. Nota: Cross-collega passi dalla parte 2 può essere usato qui. </li><li> Cellule Passage 9. </li><li> Aggiungere 10.000 cellule / cm 2 per pozzi. </li><li> Aumentare la media per 100 pl per pozzetto e incubare a 37 ° C. </li></ol></li></ol>

<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>

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Uno degli aspetti integranti della biologia è l&#39;auto-organizzazione di molecole in strutture gerarchiche che svolgono un compito specifico. 13 Nel laboratorio, auto-assemblaggio dipende da numerosi fattori quali la concentrazione di sale, il pH e la durata digestione. Come si vede, un auto-organizzazione forme idrogel quando le proteine ​​solubilizzati torna a una temperatura fisiologica. L&#39;idrogel formatosi è in grado di promuovere l&#39;attaccamento cellulare e la proliferazione in vitro.<...

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><tbody><tr><td>Triton X-100&amp;nbsp;</td><td>Fisher Scientific</td><td>BP151-100</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>Sodium Deoxycholate</td><td>Sigma-Aldrich</td><td>D6750-100g</td><td>Use in hood with eye protection and gloves.</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M7506-500g</td><td>None</td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C1016-500g</td><td>None</td></tr><tr><td>DNase</td><td>Sigma-Aldrich</td><td>D5025-150KU</td><td>None</td></tr><tr><td>HCl</td><td>Sigma-Aldrich</td><td>258148-500ML</td><td>Use with eye protection and gloves.</td></tr><tr><td>Pepsin</td><td>Sigma-Aldrich</td><td>P6887-5G</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>BP359-500</td><td>Use with eye protection and gloves.</td></tr><tr><td>Genipin</td><td>Wako Chemicals</td><td>078-03021</td><td>Use in fume hood with eye protection and gloves.</td></tr><tr><td>PBS 10x</td><td>Quality Biological</td><td>119-069-151</td><td>None</td></tr><tr><td>PBS</td><td>VWR</td><td>45000-448</td><td>None</td></tr><tr><td>Filter Paper</td><td>Whatman</td><td>8519</td><td>N/A</td></tr><tr><td>Hand pump</td><td>Fisher Scientific</td><td>10-239-1</td><td>N/A</td></tr><tr><td>Graduate Beaker</td><td>VitLab</td><td>445941</td><td>N/A</td></tr><tr><td>Cryomill</td><td>SPEX</td><td>6700</td><td>Use cryogloves and eye protection.</td></tr><tr><td>Lyophilizer</td><td>FTS FlexiDry</td><td></td><td>Use gloves.</td></tr><tr><td>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

Qui vi presentiamo un metodo per stabilire più idrogel di coltura cellulare di componenti per vitro coltura cellulare del polmone in. Cominciando con sano en bloc tessuto polmonare da suini, ratti, o il mouse, il tessuto è perfuso e immerso in successive detergenti chimici per rimuovere i detriti cellulari. Confronto istologica del tessuto prima e dopo l&#39;elaborazione conferma rimozione di oltre il 95% della doppia elica colorazione del DNA e alfa galattosidasi suggerisce la maggioranza dei detriti cellulari viene rimosso. Dopo decellularization, il tessuto è liofilizzata e quindi cryomilled in una polvere. La polvere matrice viene digerito per 48 ore in una soluzione di pepsina di digestione acida e quindi neutralizzato per formare la soluzione pregel. Gelificazione della soluzione pregel può essere indotta da incubazione a 37 ° C e può essere utilizzato immediatamente dopo neutralizzazione o conservato a 4 ° C per un massimo di due settimane. I rivestimenti possono essere formati usando la soluzione pregel su una lastra non trattata per cattaccamento ell. Le celle possono essere sospesi in pregel prima autoassemblaggio per ottenere una coltura 3D, placcato sulla superficie di un gel costituito da cui le cellule possono migrare attraverso il ponteggio, o placcato sui rivestimenti. Modifiche alla strategia presentati possono influenzare la temperatura di gelificazione, la forza, o taglie frammento di proteina. Al di là di formazione idrogel, la rigidità idrogel può essere aumentata mediante genipina.

Utilizzando questo metodo, abbiamo prodotto idrogel di maiale normale, ratto, e polmoni del mouse (Figura 1). polmoni trasformati forniscono una stima rispettivamente 5 mg, 40 mg e 10 g di polvere di ECM. Una panoramica del processo è mostrato nella figura 2. visualizzazioni chiave durante il processo sono: l&#39;aspetto bianco dei polmoni dopo il risciacquo desossicolato; dopo la formazione pregel, la soluzione deve essere opaco e la soluzione deve app...

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

Questo è un metodo per creare un 3-dimensionale scaffold coltura cellulare dalla matrice extracellulare polmonare. polmone Intact è trasformato in idrogel in grado di sostenere la crescita delle cellule in tre dimensioni.

Sintonizzabile idrogel da polmonare matrice extracellulare per colture cellulari 3D

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

tunable-hydrogels-from-pulmonary-extracellular-matrix-for-3d-cell

Research

JoVE Journal

Bioengineering

11.6K Views.  Virginia Commonwealth University.  Questo è un metodo per creare un 3-dimensionale scaffold coltura cellulare dalla matrice extracellulare polmonare. polmone Intact è trasformato in idrogel in grado di sostenere la crescita delle cellule in tre dimensioni. 

Video: Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Fabbricazione di idrogel Micropatterned per i sistemi Cultura neurali utilizzando Dynamic Fotolitografia proiezione Mask

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Ricerca

Bioingegneria

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Facile manipolazione delle architetture in idrogel a base di proteine per applicazioni della coltura delle cellule

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Biochimica

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Costruzione di fogli modulare idrogel per micropatterned macro-scala 3D Cellular Architettura

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting Cellularized Costruisce Utilizzando un tessuto-specifici idrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Microtissues 3D per la terapia rigenerativa iniettabile e droga di High-throughput Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

Coltura di cellule di idrogeli ultrasottile Innova elastico come una membrana dello scantinato biomimetici per Dual

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Produzione di elastina-come proteina idrogeli per incapsulamento e Immunostaining delle cellule in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

Ceppo controllato di idrogel 3D in imaging a microscopia dal vivo

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

Gli array di idrogel consentono una maggiore produttività per gli effetti di screening dei componenti della matrice e delle terapie nei modelli tumorali 3D

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

Idrogel biostampati fototunable per sondare le risposte cellulari all'irrigidimento della ECM

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

Autore in primo piano: Comprendere le malattie polmonari croniche utilizzando idrogel fototunable stampati in 3D 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

Idrogel fotoregistrabili in bioprinting 3D per studiare l'attivazione dei fibroblasti

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