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Graphical summary
Summary
Formation of collagen gels with tunable stiffness
The required supplies and gear embrace: excessive focus collagen (e.g. rat tail collagen Corning 354249), Minimal Important Medium (MEM, 10x) (e.g. MEM 10x, 21430-020, Gibco – Life Applied sciences), Human insulin answer (e.g. I9278, Sigma-Aldrich), distilled water (e.g. dH2O, DNAse/RNase Free, 10977-035, Gibco by Life Applied sciences), 1 N NaOH, glass cowl slips or petri dishes relying on the experimental process, falcon tubes, pipettes and pipette ideas and sterile cell tradition hood. A excessive focus collagen I answer (8.34 mg/ml, rat tail collagen, 354249, Corning) is fastidiously chosen, as not all commercially-available collagen options are acceptable for formation of 3D collagen gels. For the formation of 3D collagen I gels of tunable focus and stiffness, we use a modification of a beforehand printed protocol [1].
Preparation of histological sections from collagen gels
Atomic pressure microscopy nano-characterization
However, tapping mode is most well-liked for imaging organic specimens as it’s a extra “gentle” methodology for the reason that tip oscillates and touches the pattern floor just for a brief time frame. As an illustration, the probes acceptable for contact mode are characterised by decrease resonance frequency and softer cantilevers than these for tapping mode. In consequence, this mode will be fairly harmful, however it’s most well-liked for combining imaging with mechanical characterization measurements.
Conclusions
On this paper we introduced a protocol describing the formation of 3D collagen sort I gels with tunable stiffness, the preparation of histological sections from collagen gels and the AFM-based morphological and mechanical nano-characterization of the gels. The formation of collagen gels with tunable stiffness and the characterization of such fashions provide the benefit of offering a extra physiologically-relevant surroundings for culturing cells that additionally takes into consideration cell-ECM interactions and progress in 3D, elementary parameters which can be usually uncared for in normal in vitro research. We demonstrated that AFM provides a whole non-destructive characterization of such collagen-based tradition fashions, when it comes to morphology and mechanical properties.