Methods and Protocol for Single-Cell Motility Assays under Topological Constraints.

The extracellular environment plays a crucial role in many physiological and pathological processes involving cell motility, such as metastatic invasion in cancer development, by heavily impacting the migration strategies adopted by the cells. The study of how mechanical constraints affect the dynamics of cell migration may be relevant to gain more insight into such processes, and it may prove to be a powerful tool in the hands of biologists. In this chapter, we describe the methods used to investigate the ability of neoplastic cells to migrate through narrowing, rigid microstructures upon chemoattractant stimulation.

In vivo label-free tissue histology through a microstructured imaging window.

Tissue histopathology, based on hematoxylin and eosin (H&E) staining of thin tissue slices, is the gold standard for the evaluation of the immune reaction to the implant of a biomaterial. It is based on lengthy and costly procedures that do not allow longitudinal studies. The use of non-linear excitation microscopy in vivo, largely label-free, has the potential to overcome these limitations. With this purpose, we develop and validate an implantable microstructured device for the non-linear excitation microscopy assessment of the immune reaction to an implanted biomaterial label-free. The microstructured device, shaped as a matrix of regular 3D lattices, is obtained by two-photon laser polymerization. It is subsequently implanted in the chorioallantoic membrane (CAM) of embryonated chicken eggs for 7 days to act as an intrinsic 3D reference frame for cell counting and identification. The histological analysis based on H&E images of the tissue sections sampled around the implanted microstructures is compared to non-linear excitation and confocal images to build a cell atlas that correlates the histological observations to the label-free images. In this way, we can quantify the number of cells recruited in the tissue reconstituted in the microstructures and identify granulocytes on label-free images within and outside the microstructures. Collagen and microvessels are also identified by means of second-harmonic generation and autofluorescence imaging. The analysis indicates that the tissue reaction to implanted microstructures is like the one typical of CAM healing after injury, without a massive foreign body reaction. This opens the path to the use of similar microstructures coupled to a biomaterial, to image in vivo the regenerating interface between a tissue and a biomaterial with label-free non-linear excitation microscopy. This promises to be a transformative approach, alternative to conventional histopathology, for the bioengineering and the validation of biomaterials in in vivo longitudinal studies.