Tunable Hydrogels van Pulmonary extracellulaire matrix voor 3D-Cell Culture
Summary
Dit is een methode om een 3-dimensionale celcultuur scaffold aan pulmonale extracellulaire matrix te creëren. Intact long verwerkt tot hydrogelen die de groei van cellen in drie dimensies kan ondersteunen.
Abstract
Hier presenteren we een methode voor het vaststellen van meervoudige component celkweek hydrogels voor in vitro long celkweek. Beginnend met gezonde en bloc longweefsel van varkens, rat of muis, wordt het weefsel perfusie en ondergedompeld in daaropvolgende chemische reinigingsmiddelen om de celresten te verwijderen. Histologische vergelijking van het weefsel voor en na verwerking bevestigt verwijdering van meer dan 95% van dubbelstrengs DNA en alfa- galactosidase kleuring zien dat de meerderheid van cellulaire afval wordt verwijderd. Na decellularisatie, het weefsel gevriesdroogde en vervolgens cryomilled tot een poeder. De matrix poeder wordt gedigereerd gedurende 48 uur in een zure pepsine oplossing en daarna geneutraliseerd tot de pre-gel te vormen. Gelering van het pre-gel oplossing kan worden geïnduceerd door incubatie bij 37 ° C en kan worden gebruikt direct na neutralisatie of maximaal twee weken opgeslagen bij 4 ° C. Coatings kunnen worden gevormd met de pre-gel oplossing op niet-behandelde dienblad cell attachment. Cellen kunnen worden gesuspendeerd in de pre-gel voorafgaand aan zelfassemblage een 3D cultuur bereiken, uitgeplaat op het oppervlak van een gel gevormd waarvan de cellen kunnen migreren door het schavot, of bedekt op de bekledingen. Wijzigingen in de strategie gepresenteerd van invloed kan zijn geleringstemperatuur, kracht, of eiwit fragment maten. Beyond hydrogel formatie, kan de hydrogel stijfheid verhoogd door toepassing genipin.
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
Een van de integrale aspecten van de biologie is de zelforganisatie van moleculen in hiërarchische structuren die een specifieke taak uit te voeren. 13 In het laboratorium zelfassemblage afhankelijk van talrijke factoren zoals zoutconcentratie, pH en duur spijsvertering. Zoals getoond zelforganiserend hydrogel vormen wanneer opgeloste eiwitten weer een fysiologische temperatuur. De gevormde hydrogel kan bevorderen cellulaire hechting en proliferatie in vitro.
Cellulaire …
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
We willen graag Smithfield boerderijen bedanken voor het doneren van het intacte varkens longweefsel. We willen ook graag bedanken Dr. Hu Yang, Dr. Christina Tang en de afdeling Heelkunde VCU Plastic voor het mogelijk maken om hun apparatuur te gebruiken. Hydrogel en weefselmonsters werden voorbereid voor SEM bij de VCU Afdeling Anatomie en Neurobiologie Microscopy Facility ondersteund, voor een deel, door de financiering van NIH-NINDS Center Core Grant 5 P30 NS047463 en, gedeeltelijk, door de financiering vorm NIH-NCI Cancer Center Ondersteuning Grant P30 CA016059. SEM beeldvorming van monsters bij de VCU Nanotechnologie Core karakterisering Facility (NCC). Dit werk werd gefinancierd door de 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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