The Isolated Mucosa of the Rat Colon Decellularization, Microscopy and Cell Cultures.

This project presents the most important findings of the studies, which we carried out in our laboratory on the decellularization of the rat isolated colonic mucosa. We have also included some details of the experiences gathered with the muscle layer as well as the whole wall of the colon. The question of the cytocompatibility of this new substrate has been addressed with the application of primary cultures of human cells and well-established cell lines. The possible applications in experimental and medical settings will be discussed.

The isolated colonic mucosa: a new substrate for tissue reconstruction. Isolation, decellularization and characterization by immunohistochemistry and electron microscopy

This study proposes the use of isolated colonic mucosa as a "scaffold" for cell cultures and potentially for tissue reconstruction. The main goal of this study was to obtain complete decellularization of the specimens while preserving the superficial basement membrane (BM) as a place for cell attachment and growth. This decellularization technique uses a chelating agent in combination with mechanical vibration, followed by detergents and DNase. The grade of decellularization achieved is assessed by counting the number of cell nuclei stained with propidium iodide (PI). BM marker proteins such as collagen IV, laminin and perlecan were detected by immunohistochemistry (ICC). Transmission (TEM) and scanning electron microscopy (SEM) were used to examine the BM ultrastructure and surface topography. The results show that the protocol used is suitable for rat colonic mucosa. During the process, material of the lamina lucida (LL) was partly removed from the BM, whereas the lamina densa (LD) seems to have remained unchanged. The BM had become thinner than the control specimens. The nanotopography of the BM surface is characterized by globules of 34-60 nm in diameter. Human fetal fibroblasts were successfully cultured on this substrate confirming that cells can adhere to and grow on this substrate, at least for the particular cell line used. It can be said that the colonic mucosa is an interesting substrate for in vitro stud-ies with cells and presumably also for tissue reconstruction

No disponible

Morphological studies on platelet function using a microfluidic chip. A proof of concept study

Platelets are blood cellular components involved in hemostatic processes and thrombus formation. Activation and inhibition of platelets result in an increase in morphological changes and a significant reduction in adhesion. There are several approaches towards the determination of the functional status of platelets, based on criteria such as cell adhesion, molecular changes at the cell surface, etc. In recent years, microfluidic devices have been introduced to mimic conditions proper to the vascular system, and so emulate thrombus formation in vivo. This study presents a microchip, the Thrombi Chip® , which is partially fitted with fluidic properties. This microchip has various types of micro-channels into which the platelets are inserted and, after drug treatment, the investigation is completed with the examination of the chip under an invert light microscope. For microscopy, cells were labeled with FCDA (human platelets) and Rho6G (mouse platelets). Counts and morphometric measurements of the adhered cells were carried out using digital images. To validate the results obtained with the microchip, the fractions of mice platelets were investigated with flow cytometry as well. Scanning electron microscopy was used to examine the morphological changes related to activation and inhibition in human platelets. The results show that, with this microchip, activation and inhibition of platelets can be detected. Flow cytometry studies largely confirm the microchip results. Certain variability in the results observed in human platelets is considered normal, as donors were randomized. In this respect the mouse platelets were much more uniform. Measurements with the microchip require that the sample be divided into three groups: control, activated and inhibited, resulting in a set of data, which, after respective evaluation, provides activity profiles, giving information on the status and response capacity of a sample. Such profiles could have diagnostic relevance and therefore be useful in a clinical context, for example in the monitoring of the effects of short- and long-term treatment of patients, as well as to test new drugs

No disponible