In vivo imaging system for explants analysis-A new approach for assessment of cell transplantation effects in large animal models

PLoS One. 2017 Sep 20;12(9):e0184588. doi: 10.1371/journal.pone.0184588. eCollection 2017.


Introduction: Despite spectacular progress in cellular transplantology, there are still many concerns about the fate of transplanted cells. More preclinical studies are needed, especially on large animal models, to bridge the translational gap between basic research and the clinic. Herein, we propose a novel approach in analysis of cell transplantation effects in large animals explants using in vivo imaging system (IVIS®) or similar equipment.

Material and methods: In the in vitro experiment cells labeled with fluorescent membrane dyes: DID (far red) or PKH26 (orange) were visualized with IVIS®. The correlation between the fluorescence signal and cell number with or without addition of minced muscle tissue was calculated. In the ex vivo study urethras obtained from goats after intraurethral cells (n = 9) or PBS (n = 4) injections were divided into 0.5 cm cross-slices and analyzed by using IVIS®. Automatic algorithm followed or not by manual setup was used to separate specific dye signal from tissue autofluorescence. The results were verified by systematic microscopic analysis of standard 10 μm specimens prepared from slices before and after immunohistochemical staining. Comparison of obtained data was performed using diagnostic test function.

Results: Fluorescence signal strength in IVIS® was directly proportional to the number of cells regardless of the dye used and detectable for minimum 0.25x106 of cells. DID-derived signal was much less affected by the background signal in comparison to PKH26 in in vitro test. Using the IVIS® to scan explants in defined arrangement resulted in precise localization of DID but not PKH26 positive spots. Microscopic analysis of histological specimens confirmed the specificity (89%) and sensitivity (80%) of IVIS® assessment relative to DID dye. The procedure enabled successful immunohistochemical staining of specimens derived from analyzed slices.

Conclusions: The IVIS® system under appropriate conditions of visualization and analysis can be used as a method for ex vivo evaluation of cell transplantation effects. Presented protocol allows for evaluation of cell delivery precision rate, enables semi-quantitative assessment of signal, preselects material for further analysis without interfering with the tissue properties. Far red dyes are appropriate fluorophores to cell labeling for this application.

MeSH terms

  • Animals
  • Cell Proliferation
  • Cell Tracking / methods*
  • Cells, Cultured
  • Female
  • Fluorescence
  • Fluorescent Dyes / metabolism*
  • Goats
  • Mesenchymal Stem Cell Transplantation*
  • Mesenchymal Stem Cells / cytology*
  • Molecular Imaging / methods*
  • Organic Chemicals / metabolism
  • Urethra / cytology
  • Urethra / metabolism*


  • Fluorescent Dyes
  • Organic Chemicals
  • PKH 26

Grant support

This study was supported within Parent Bridge program of the Foundation for Polish Science, co-financed by the European Union under the European Regional Development Fund. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.