Paraffin Embedding Allows Effective Analysis of Proliferation, Survival, and Immunophenotyping of Cells Cultured on Poly(l-Lactic Acid) Electrospun Nanofiber Scaffolds
- 1 August 2010
- journal article
- research article
- Published by Mary Ann Liebert Inc in Tissue Engineering, Part C: Methods
- Vol. 16 (4), 751-760
- https://doi.org/10.1089/ten.tec.2009.0316
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.Keywords
This publication has 33 references indexed in Scilit:
- Mesenchymal stem cell interaction with a non‐woven hyaluronan‐based scaffold suitable for tissue repairJournal of Anatomy, 2008
- Functional Hepatocyte-Like Cells Derived from Human Bone Marrow Mesenchymal Stem Cells on a Novel 3-Dimensional Biocompatible Nanofibrous ScaffoldThe International Journal of Artificial Organs, 2008
- Osteoblastic Phenotype Expression of MC3T3-E1 Cultured on Electrospun Polycaprolactone Fiber Mats Filled with Hydroxyapatite NanoparticlesBiomacromolecules, 2007
- In vitro evaluation of electrospun nanofiber scaffolds for vascular graft applicationJournal of Biomedical Materials Research Part A, 2007
- Esophageal epithelium regeneration on fibronectin grafted poly(l-lactide-co-caprolactone) (PLLC) nanofiber scaffoldBiomaterials, 2007
- The Role of Electrospinning in the Emerging Field of NanomedicineCurrent Pharmaceutical Design, 2006
- Growth of Mesenchymal Stem Cells on Electrospun Type I Collagen NanofibersThe International Journal of Cell Cloning, 2006
- Gravity spun polycaprolactone fibres for soft tissue engineering: Interaction with fibroblasts and myoblasts in cell cultureBiomaterials, 2006
- Effect of fiber diameter on spreading, proliferation, and differentiation of osteoblastic cells on electrospun poly(lactic acid) substratesBiomaterials, 2005
- The development of an embedding technique for polylactide spongesJournal of Biomedical Materials Research, 1999