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Characterization of sample preparation methods of NIH/3T3 fibroblasts for ToF-SIMS analysis.

ABSTRACT: The information that is obtained from single cells during time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis is influenced by the method that was used to prepare the cells. The removal of extracellular media before analysis is necessary, but the rinsing technique should not damage the plasma membrane of the cell. The presence of intracellular salts reduced the secondary ion yield an average of 2.6-fold during Bi3 (+)/C60 (++) depth profiles. Chemical fixation followed by rinsing removed a majority of the intracellular salts, "recovering" the positive secondary ion yields. The formaldehyde-fixation process removed a majority of the intracellular Cl(-), but other key anions were not removed in significant amounts. The data presented here is consistent the anion neutralization mechanism largely responsible for the lower ion yields. All of the organic secondary ions that were detected in the freeze-dried cells were also detected in the formaldehyde-fixed cells, suggesting that the fixation process did not remove any molecular species to an extent that is detectable by ToF-SIMS. Compared to freeze dried cells, well preserved, frozen-hydrated cells showed little increase, or a decreased yield, for most low mass ions, but an increased yield for larger mass fragments. This is consistent with a reduced damage cross section at cryogenic analysis temperatures, although proton donation from water and reduction the salt effects in the presence of water likely also play roles. Numerous ions detected from the frozen-hydrated cells were not detected from the freeze dried cells, however many of these ions were attributed to chemical combinations of water, salts and the ammonium acetate rinsing solution.

SUBMITTER: Robinson MA

PROVIDER: S-EPMC4000548 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundSeveral materials have been used for tissue engineering purposes, since the ideal matrix depends on the desired tissue. Silk biomaterials have come to focus due to their great mechanical properties. As untreated silkworm silk has been found to be quite immunogenic, an alternative could be spider silk. Not only does it own unique mechanical properties, its biocompatibility has been shown already in vivo. In our study, we used native spider dragline silk which is known as the strongest fibre in nature.Methodology/principal findingsSteel frames were originally designed and manufactured and woven with spider silk, harvesting dragline silk directly out of the animal. After sterilization, scaffolds were seeded with fibroblasts to analyse cell proliferation and adhesion. Analysis of cell morphology and actin filament alignment clearly revealed adherence. Proliferation was measured by cell count as well as determination of relative fluorescence each after 1, 2, 3, and 5 days. Cell counts for native spider silk were also compared with those for trypsin-digested spider silk. Spider silk specimens displayed less proliferation than collagen- and fibronectin-coated cover slips, enzymatic treatment reduced adhesion and proliferation rates tendentially though not significantly. Nevertheless, proliferation could be proven with high significance (p<0.01).Conclusion/significanceNative spider silk does not require any modification to its application as a biomaterial that can rival any artificial material in terms of cell growth promoting properties. We could show adhesion mechanics on intracellular level. Additionally, proliferation kinetics were higher than in enzymatically digested controls, indicating that spider silk does not require modification. Recent findings concerning reduction of cell proliferation after exposure could not be met. As biotechnological production of the hierarchical composition of native spider silk fibres is still a challenge, our study has a pioneer role in researching cellular mechanics on native spider silk fibres.

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Characterization of sample preparation methods of NIH/3T3 fibroblasts for ToF-SIMS analysis.

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