A new holistic 3D non-invasive analysis of cellular distribution and motility on fibroin-alginate microcarriers using light sheet fluorescent microscopy

Serena Duchi, Filippo Piccinini, Michela Pierini, Alessandro Bevilacqua, Maria Luisa Torre, Enrico Lucarelli, Spartaco Santi

Research output: Contribution to journalArticle

Abstract

Cell interaction with biomaterials is one of the keystones to developing medical devices for tissue engineering applications. Biomaterials are the scaffolds that give three-dimensional support to the cells, and are vectors that deliver the cells to the injured tissue requiring repair. Features of biomaterials can influence the behaviour of the cells and consequently the efficacy of the tissue-engineered product. The adhesion, distribution and motility of the seeded cells onto the scaffold represent key aspects, and must be evaluated in vitro during the product development, especially when the efficacy of a specific tissue-engineered product depends on viable and functional cell loading. In this work, we propose a non-invasive and non-destructive imaging analysis for investigating motility, viability and distribution of Mesenchymal Stem Cells (MSCs) on silk fibroin-based alginate microcarriers, to test the adhesion capacity of the fibroin coating onto alginate which is known to be unsuitable for cell adhesion. However, in depth characterization of the biomaterial is beyond the scope of this paper. Scaffold-loaded MSCs were stained with Calcein-AM and Ethidium homodimer-1 to detect live and dead cells, respectively, and counterstained with Hoechst to label cell nuclei. Time-lapse Light Sheet Fluorescent Microscopy (LSFM) was then used to produce three-dimensional images of the entire cells-loaded fibroin/alginate microcarriers. In order to quantitatively track the cell motility over time, we also developed an open source user friendly software tool called Fluorescent Cell Tracker in Three-Dimensions (F-Tracker3D). Combining LSFM with F-Tracker3D we were able for the first time to assess the distribution and motility of stem cells in a non-invasive, non-destructive, quantitative, and three-dimensional analysis of the entire surface of the cell-loaded scaffold. We therefore propose this imaging technique as an innovative holistic tool for monitoring cell-biomaterial interactions, and as a tool for the design, fabrication and functionalization of a scaffold as a medical device.

Original languageEnglish
Pages (from-to)e0183336
JournalPLoS One
Volume12
Issue number8
DOIs
Publication statusPublished - 2017

Fingerprint

Fibroins
fibroins
Biocompatible Materials
alginates
Microscopy
microscopy
Microscopic examination
Scaffolds
Light
biocompatible materials
Stem cells
Tissue
cells
Adhesion
Cells
medical equipment
stem cells
Imaging techniques
Mesenchymal Stromal Cells
Cell Communication

Keywords

  • Alginates
  • Cell Adhesion
  • Fibroins
  • Glucuronic Acid
  • Hexuronic Acids
  • Humans
  • Mesenchymal Stromal Cells
  • Microscopy, Fluorescence
  • Tissue Engineering
  • Journal Article

Cite this

A new holistic 3D non-invasive analysis of cellular distribution and motility on fibroin-alginate microcarriers using light sheet fluorescent microscopy. / Duchi, Serena; Piccinini, Filippo; Pierini, Michela; Bevilacqua, Alessandro; Torre, Maria Luisa; Lucarelli, Enrico; Santi, Spartaco.

In: PLoS One, Vol. 12, No. 8, 2017, p. e0183336.

Research output: Contribution to journalArticle

Duchi, Serena ; Piccinini, Filippo ; Pierini, Michela ; Bevilacqua, Alessandro ; Torre, Maria Luisa ; Lucarelli, Enrico ; Santi, Spartaco. / A new holistic 3D non-invasive analysis of cellular distribution and motility on fibroin-alginate microcarriers using light sheet fluorescent microscopy. In: PLoS One. 2017 ; Vol. 12, No. 8. pp. e0183336.
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