Fleksibel Hydrogeler fra Lunge ekstracellulær matriks for 3D Cell Culture
Summary
Dette er en metode for å lage en tre-dimensjonal cellekultur stillaset fra lunge ekstracellulære matriks. Intakt lunge blir bearbeidet til hydrogeler som kan understøtte veksten av celler i tre dimensjoner.
Abstract
Her presenterer vi en fremgangsmåte for å etablere flere komponentcellekultur hydrogeler for in vitro lungecellekultur. Fra og med sunn en bloc lungevev fra svin, rotte eller mus, er vevet perfused og nedsenket i påfølgende kjemiske vaskemidler for å fjerne cellerester. Histologisk sammenligning av vev før og etter behandling bekrefter fjernet over 95% av dobbeltkjedet DNA og alfa-galaktosidase farging antyder de fleste av cellerester fjernes. Etter decellularization, er vevet lyofilisert og deretter cryomilled til et pulver. Matrisen pulver oppløses i 48 timer i en sur løsning fordøyelsen pepsin og deretter nøytraliseres til å danne det Pregel oppløsning. Gelering av Pregel oppløsningen kan induseres ved inkubering ved 37 ° C og kan brukes umiddelbart etter nøytralisering eller lagret ved 4 ° C i opptil to uker. Belegg kan dannes ved hjelp av Pregel løsning på en ubehandlet plate for cell vedlegg. Celler kan bli suspendert i Pregel før selv-montering for å oppnå en 3D-kultur, belagt på overflaten av en formet gel hvorfra cellene kan migrere inn i stillaset, eller belagt på beleggene. Endringer i strategien presenteres kan påvirke geleringstemperaturen, styrke eller proteinfragmentstørrelser. Utover hydrogel formasjon, kan hydrogelen stivhet økes ved å bruke 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
En av de integrerte aspekter av biologi er selvorganisering av molekyler i hierarkiske strukturer som utfører en bestemt oppgave. 13 I laboratoriet, avhenger selv-sammenstillingen på en rekke faktorer som for eksempel saltkonsentrasjon, pH-verdi, og fordøyelse varighet. Som vist, et selvorganiserende hydrogel dannes når solubiliserte proteiner som går tilbake til et fysiologisk temperatur. Den hydrogel som dannes er i stand til å fremme cellulær festing og formering in vitro.
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The authors have nothing to disclose.
Acknowledgements
Vi vil gjerne takke Smith gårder for å donere intakt svin lungevevet. Vi ønsker også å takke Dr. Hu Yang, Dr. Christina Tang og VCU plastisk kirurgi Avdeling for å tillate oss å bruke deres utstyr. Hydrogel og vevsprøver var forberedt på SEM ved VCU Institutt for anatomi og nevrobiologi Mikros Facility støttes delvis av midler fra NIH-ninds Senter Kjerne Grant 5 P30 NS047463 og dels av midler skjema NIH-NCI Cancer Center Support Grant P30 CA016059. SEM avbildning av prøvene på VCU Nanoteknologi Kjerne Karakterisering Facility (NCC). Dette arbeidet ble finansiert av 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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