Sintonizzabile idrogel da polmonare matrice extracellulare per colture cellulari 3D
Summary
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.
Abstract
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'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.
Introduction
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.
Protocol
Representative Results
Discussion
Uno degli aspetti integranti della biologia è l'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'idrogel formatosi è in grado di promuovere l'attaccamento cellulare e la proliferazione in vitro.<…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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'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.
Materials
| Triton X-100 | Fisher Scientific | BP151-100 | Use in fume hood with eye protection and gloves. |
| Sodium Deoxycholate | Sigma-Aldrich | D6750-100g | Use with eye protection and gloves. |
| Magnesium Sulfate | Sigma-Aldrich | M7506-500g | None |
| Calcium Chloride | Sigma-Aldrich | C1016-500g | None |
| DNase | Sigma-Aldrich | D5025-150KU | None |
| HCl | Sigma-Aldrich | 258148-500ML | Use with eye protection and gloves. |
| Pepsin | Sigma-Aldrich | P6887-5G | Use in fume hood with eye protection and gloves. |
| Sodium Hydroxide | Fisher Scientific | BP359-500 | Use with eye protection and gloves. |
| Genipin | Wako Chemicals | 078-03021 | Use in fume hood with eye protection and gloves. |
| PBS 10x | Quality Biological | 119-069-151 | None |
| PBS | VWR | 45000-448 | None |
| Filter Paper | Whatman | 8519 | N/A |
| Hand pump | Fisher Scientific | 10-239-1 | N/A |
| Graduate Beaker | VitLab | 445941 | N/A |
| Cryomill | SPEX | 6700 | Use cryogloves and eye protection. |
| Lyophilizer | FTS FlexiDry | Use gloves. | |
| Rheometer | Discovery | HR-2 | Use gloves and eye protection. |
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