Tunable Hydrogeler fra Pulmonal ekstracellulære matrix for 3D cellekultur
Summary
Dette er en metode til at skabe en 3-dimensionel cellekultur stillads fra pulmonal ekstracellulære matrix. Intakt lunge forarbejdes til hydrogeler, der kan understøtte væksten af celler i tre dimensioner.
Abstract
Her præsenteres en fremgangsmåde til etablering af flere komponenter cellekultur hydrogeler til in vitro lunge-cellekultur. Begyndende med sund en bloc lungevæv fra svin, rotte eller mus, er vævet perfunderes og nedsænket i efterfølgende kemiske rengøringsmidler til fjernelse af cellerester. Histologisk sammenligning af vævet før og efter forarbejdning bekræfter fjernelse af over 95% af dobbeltstrenget DNA og alfa galactosidase-farvning antyder størstedelen af cellerester fjernes. Efter decellularization, vævet er lyofiliseret og derefter cryomilled til pulver. Matricen pulver fordøjes i 48 timer i et surt pepsinfordøjelse opløsning og derefter neutraliseret til dannelse prægelen opløsning. Gelering af prægelen opløsning kan induceres ved inkubation ved 37 ° C og kan anvendes umiddelbart efter neutralisering eller opbevaret ved 4 ° C i op til to uger. Belægninger kan dannes under anvendelse af prægelen løsning på et ikke-behandlede plade til cell vedhæftet fil. Celler kan suspenderes i før selv-samling for at opnå en 3D kultur prægelen, udpladet på overfladen af en dannet gel fra hvilken cellerne kan migrere gennem stilladset, eller udpladet på belægningerne. Ændringer den strategi, kan påvirke geleringstemperaturen, styrke, eller proteinfragment størrelser. Beyond hydrogel-dannelse, kan hydrogelen stivhed forøges ved anvendelse 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
Et af de integrerede aspekter af biologi er selvorganisering af molekyler i hierarkiske strukturer, der udfører en bestemt opgave. 13 I laboratoriet, saml-selv afhænger af mange faktorer såsom saltkoncentration, pH, og fordøjelse varighed. Som vist, et selvorganiserende hydrogel former når solubiliserede proteiner vender tilbage til en fysiologisk temperatur. Hydrogelen dannes, er i stand til at fremme cellulær vedhæftning og proliferation in vitro.
Cellulære resp…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Vi vil gerne takke Smithfield gårde for at donere den intakte porcint lungevæv. Vi vil også gerne takke Dr. Hu Yang, Dr. Christina Tang og VCU plastikkirurgi Institut for at tillade os at bruge deres udstyr. Hydrogel og vævsprøver blev forberedt SEM ved VCU Anatomisk Institut og Neurobiologi Microscopy Facility understøttes til dels af finansiering fra NIH-NINDS center Core Grant 5 P30 NS047463 og dels ved finansiering formular NIH-NCI Cancer Center Support Grant P30 CA016059. SEM billeddannelse af prøver på VCU Nanoteknologi Core Karakterisering Facility (NCC). Dette arbejde blev finansieret af 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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