Paraffin Embedding Allows Effective Analysis of Proliferation, Survival, and Immunophenotyping of Cells Cultured on Poly(l-Lactic Acid) Electrospun Nanofiber Scaffolds

Abstract

Morphological and immunophenotypic characterization of cells grown on poly(l-lactic acid) (PLLA) electrospun scaffolds is usually performed using immunofluorescence and cryosections. However, these methods present practical limits; histological processing, on the other hand, is believed to lead to artifactual changes in the scaffold structure. Here the formalin-fixed paraffin-embedding (FFPE) procedure was tailored to process PLLA electrospun scaffolds grown with human umbilical vein endothelial cells. After 1 to 7 days of culture, the scaffolds were processed with the FFPE procedure. Using this protocol, not only cross sections but also "en face" sections were obtained. This made possible to perform the effective light microscopy analysis of cell morphology and to assess cell adhesion and penetration without considerable scaffold damage. The method was also suitable for immunohistochemical assays, such as proliferation (Ki67), extracellular matrix production (type IV collagen), survival (cleaved caspase-3), and immunophenotyping (KDR, CD44, vimentin, CD45); results were compared with those obtained using complementary techniques (scanning electron microscopy, Alamar Blue assay, and cryosections). The FFPE protocol can be safely applied to PLLA scaffolds and provides information that are essential to study the mechanisms of interaction between cells and PLLA fibers before their potential implantation in vivo.