细胞生物学

The ex vivo experimentation with surgically discarded human skin represents a unique methodology amenable for mechanism and pharmacologic agent studies without the involvement of human subjects. Here, we describe a protocol that includes preparation, culture, and stimulation of human skin explants, and subsequent analyses by quantitative reverse transcription PCR and immunostaining. This protocol may also be applied for ex vivo studies of murine skin, reducing animal numbers and potentially harmful treatments. In our hands, this protocol has been used for wound healing, viral infection, and hair growth–related studies.

Graphical abstract:

Cartoon of explant skin culture. Skin explant sits on top of a gelatin surgical sponge saturated with culture medium at an air–liquid interface.

Enteroendocrine cells (EECs) are known chemosensors in the gastrointestinal (GI) epithelium. They release a diversity of gut hormones in response to various stimuli. Here, we report an in-vitro assay to measure GLP-1 release from cultured murine EEC’s under fatty acid stimulation.

Slices of neuronal tissue maintain a high degree of topographical and functional properties of neurons and glia and therefore are extensively used for measurements of neuronal activity at the molecular, cellular and network levels. However, the lifespan of slice preparations is narrow, averaging of 6-8 hours. Moreover, the average viability of brain slices varies according to animal age and region of interest, leading to the high variability and low reproducibility of recorded data.

Previous techniques to increase the viability of brain slices focused on reducing cytotoxicity by chemical means, including alterations of the artificial cerebrospinal fluid (aCSF) composition to alleviate the direct damage of the slicing procedure or adding protective antioxidants to reduce cellular deterioration. In this protocol, we use a combination of hypothermia with firm control of the aCSF conditions in the recovery chamber (pH, temperature, and bacteria levels) to extend the slice viability significantly.

Given the breadth of its usage, improving slice viability and longevity can considerably increase data reproducibility and reduce the cost, time, and number of animals used in neurophysiological studies.

Cells generate mechanical forces to shape tissues during morphogenesis. These forces can activate several biochemical pathways and trigger diverse cellular responses by mechano-sensation, such as differentiation, division, migration and apoptosis. Assessing the mechano-responses of cells in living organisms requires tools to apply controlled local forces within biological tissues. For this, we have set up a method to generate controlled forces on a magnetic particle embedded within a chosen tissue of Drosophila embryos. We designed a protocol to inject an individual particle in early embryos and to position it, using a permanent magnet, within the tissue of our choice. Controlled forces in the range of pico to nanonewtons can be applied on the particle with the use of an electromagnet that has been previously calibrated. The bead displacement and the epithelial deformation upon force application can be followed with live imaging and further analyzed using simple analysis tools. This method has been successfully used to identify changes in mechanics in the blastoderm before gastrulation. This protocol provides the details, (i) for injecting a magnetic particle in Drosophila embryos, (ii) for calibrating an electromagnet and (iii) to apply controlled forces in living tissues.

To assess oncogenic potential, classical transformation assays are based on cell line models. However, cell line based models do not reflect the complexity of human tissues. We thus developed an inducible expression system for gene expression in ex vivo human tissues, which maintain native tissue architecture, such as epithelia and stroma. To validate the system, we transduced and expressed known tumor suppressors (p53, p33ING1b), oncoproteins (RasV12, p47ING3), or controls (empty vector, YFP) in ex vivo prostate tissues, then assessed proliferation by immunohistochemistry of markers (H3S10^phos). Herein, we describe how to generate lentiviral vectors and particules, successfully transduce human prostate tissues, induce exogenous gene expression, and assess cellular proliferation.