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

Zarychta-Wiśniewska, Weronika; Burdzinska, Anna; Zagozdzon, Radosław; Dybowski, Bartosz; Butrym, Marta LU ; Gajewski, Zdzisław and Paczek, Leszek (2017) In PLoS ONE 12(9).
Abstract

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... (More)

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.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
in vivo imaging, cell transplantation, Animal model
in
PLoS ONE
volume
12
issue
9
publisher
Public Library of Science
external identifiers
  • scopus:85029703408
  • pmid:28931067
  • wos:000411314700023
ISSN
1932-6203
DOI
10.1371/journal.pone.0184588
language
English
LU publication?
yes
id
4f25814a-fd2a-4d29-9186-2d7abbc7d894
date added to LUP
2017-10-10 11:34:51
date last changed
2018-01-16 13:22:28
@article{4f25814a-fd2a-4d29-9186-2d7abbc7d894,
  abstract     = {<p>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 0.25x10<sup>6</sup> 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.</p>},
  articleno    = {e0184588},
  author       = {Zarychta-Wiśniewska, Weronika and Burdzinska, Anna and Zagozdzon, Radosław and Dybowski, Bartosz and Butrym, Marta and Gajewski, Zdzisław and Paczek, Leszek},
  issn         = {1932-6203},
  keyword      = {in vivo imaging,cell transplantation,Animal model},
  language     = {eng},
  month        = {09},
  number       = {9},
  publisher    = {Public Library of Science},
  series       = {PLoS ONE},
  title        = {In vivo imaging system for explants analysis—A new approach for assessment of cell transplantation effects in large animal models},
  url          = {http://dx.doi.org/10.1371/journal.pone.0184588},
  volume       = {12},
  year         = {2017},
}