European Journal of Anatomy

Official Journal of The Spanish Society of Anatomy
Cover Volume 22 - Number 1
Eur J Anat, 22 (1): 1-15 (2018)

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

Laura López-Gómez1, Teresa Núñez-López2, Gilberto del Rosario-Hernández2, Pedro Mestres-Ventura1, 3

1Department of Human Histology and Pathology, Faculty for Health Sciences, University Rey Juan Carlos , Av. de Atenas s/n, E-28922 Alcorcón, Madrid, Spain, 2Laboratory of Electron Microscopy, Centre for Technical Support (CAT), University Rey Juan Carlos, 28933 Mostoles, Madrid, Spain, 3Department of Anatomy and Cell Biology, Medi-cal School, Saarland University, 66421 Homburg (Germany)

ABSTRACT 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 at-tachment 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 charac-terized 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.

Keywords: Scaffold – Colonic mucosa – Base-ment membrane – Decellularization – Electron mi-croscopy – Immunohistochemistry

European Journal of anatomy
ISSN 2340-311X (Online)