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3D polyelectrolyte scaffolds to mimic exocrine glands: a step towards a prostate-on-chip platform

Nathalie Picollet-D’hahan
Nathalie Picollet-D’hahan
, 
Axel Tollance
Axel Tollance
, 
Cristina Belda Marin
Cristina Belda Marin
, 
Lavinia Liguori
Lavinia Liguori
, 
Christophe Marquette
Christophe Marquette
, 
Odile Filhol-Cochet
Odile Filhol-Cochet
, 
Isabelle Vilgrain
Isabelle Vilgrain
, 
Guillaume Laffitte
Guillaume Laffitte
, 
Florence Rivera
Florence Rivera
, 
Jean-Pierre Alcaraz
Jean-Pierre Alcaraz
, 
Jacques Thélu
Jacques Thélu
, 
Olivier Nicoud
Olivier Nicoud
, 
Thibaud Moufle-Milot
Thibaud Moufle-Milot
, 
Maxime Legues
Maxime Legues
, 
Ali Bouamrani
Ali Bouamrani
, 
Adrien Mombrun
Adrien Mombrun
, 
Benoit Gilquin
Benoit Gilquin
, 
Sophie Gerbaud
Sophie Gerbaud
, 
Patricia Obeid
Patricia Obeid
, 
Fréderique Kermarrec
Fréderique Kermarrec
, 
Xavier Gidrol
Xavier Gidrol
 e 
Donald K. Martin
Donald K. Martin
   | 27 ott 2018
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Article Category: Research Article
Pubblicato online: 27 ott 2018
Pagine: 180 - 191
DOI: https://doi.org/10.2478/ebtj-2018-0048
Parole chiave
© 2018 Nathalie Picollet-D’hahan, Axel Tollance, Cristina Belda Marin, Lavinia Liguori, Christophe Marquette, Odile Filhol-Cochet, Isabelle Vilgrain, Guillaume Laffitte, Florence Rivera, Jean-Pierre Alcaraz, Jacques Thélu, Olivier Nicoud, Thibaud Moufle-Milot, Maxime Legues, Ali Bouamrani, Adrien Mombrun, Benoit Gilquin, Sophie Gerbaud, Patricia Obeid, Fréderique Kermarrec, Xavier Gidrol, Donald K. Martin, published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Figure 1

Mould design and fabrication. A. The Master mould was obtained by dropping a biocompatible resin on a rigid resin support. The PDMS final mould is composed by 200 hemi-spherical structures of ~ 100 μm on the base and ~ 50 um in heights. The pitch between the hemispherical wells is ~ 400 μm. B. Scanning electron microscopy (SEM) of the surface and the hemispheres. C. Confocal microscopy (orthogonal section) of the hemisphere coated with multi PE (15 layers alternating PSS-PAH) incubated with fluorescein. The fluorescein labels PE allowing the thin layer visualization. D. Confocal scanning laser microscopy of the hemispheres.
Mould design and fabrication. A. The Master mould was obtained by dropping a biocompatible resin on a rigid resin support. The PDMS final mould is composed by 200 hemi-spherical structures of ~ 100 μm on the base and ~ 50 um in heights. The pitch between the hemispherical wells is ~ 400 μm. B. Scanning electron microscopy (SEM) of the surface and the hemispheres. C. Confocal microscopy (orthogonal section) of the hemisphere coated with multi PE (15 layers alternating PSS-PAH) incubated with fluorescein. The fluorescein labels PE allowing the thin layer visualization. D. Confocal scanning laser microscopy of the hemispheres.

Figure 2

Conceptual view of a device integrating nanostructured free-standing supporting membrane. A. Schematic architecture of finger-like nanostructured scaffolds. The polyelectrolyte membrane (PEM, in red) creates acinar/ductal free standing and porous 3D structures that enclose the top of the microfluidic culture chamber to control the microenvironment for the cells. B. Close-up of the porous nanostructured membrane. Epithelial cells (EC, in green) are on the inside of the polyelectrolyte membrane. Cancerous cells are spiked from the outside of the polyelectrolyte membrane. Arrows indicate the secretions produced by EC. C. Schematic process of finger-like PE fabrication with sacrificial core (in purple).
Conceptual view of a device integrating nanostructured free-standing supporting membrane. A. Schematic architecture of finger-like nanostructured scaffolds. The polyelectrolyte membrane (PEM, in red) creates acinar/ductal free standing and porous 3D structures that enclose the top of the microfluidic culture chamber to control the microenvironment for the cells. B. Close-up of the porous nanostructured membrane. Epithelial cells (EC, in green) are on the inside of the polyelectrolyte membrane. Cancerous cells are spiked from the outside of the polyelectrolyte membrane. Arrows indicate the secretions produced by EC. C. Schematic process of finger-like PE fabrication with sacrificial core (in purple).

Figure 3

Schematic of alginate gelation process over 3D printed moulds. A. sacrificial alginate mould (c) is created over a 3D printed piece (a) to obtain a 3D shaped polyelectrolyte membrane (d). B. List of the 3D printed pieces. The assembly of these pieces allow the creation of PE pillar-like structures.
Schematic of alginate gelation process over 3D printed moulds. A. sacrificial alginate mould (c) is created over a 3D printed piece (a) to obtain a 3D shaped polyelectrolyte membrane (d). B. List of the 3D printed pieces. The assembly of these pieces allow the creation of PE pillar-like structures.

Figure 4

Hemisphere-shaped PE scaffolds. A. RWPE-1 cells cultured for 5 days on a PE coated mould. A deep color elaboration of the 3D acquisition shows the cellular distribution on the coated hemisphere (Red 0 μm- blue 50 μm). B. Calibration curve to determine the volume of secretion. C. Elispot detection of secretions from WPE1-int cells w/o DHT (a); WPE1-int w/DHT (b); RPTEC cells (negative control) (c) and 3 ng pure PSA (positive control) (d).
Hemisphere-shaped PE scaffolds. A. RWPE-1 cells cultured for 5 days on a PE coated mould. A deep color elaboration of the 3D acquisition shows the cellular distribution on the coated hemisphere (Red 0 μm- blue 50 μm). B. Calibration curve to determine the volume of secretion. C. Elispot detection of secretions from WPE1-int cells w/o DHT (a); WPE1-int w/DHT (b); RPTEC cells (negative control) (c) and 3 ng pure PSA (positive control) (d).

Figure 5

MALDI profiling statistical analysis of 3 cell lines supernatant. A. Principal Component Analysis of PC3, LNCaP and PNT2 spectra. B. Unsupervised hierarchical clustering of PC3, LNCaP and PNT2 spectra. Peaks used for 2D peak distribution are highlighted in orange. C. 2D peak distribution, plotting the intensities of peaks 2425 Da and 2482 Da across PC3, LNCaP and PNT2 spectra.
MALDI profiling statistical analysis of 3 cell lines supernatant. A. Principal Component Analysis of PC3, LNCaP and PNT2 spectra. B. Unsupervised hierarchical clustering of PC3, LNCaP and PNT2 spectra. Peaks used for 2D peak distribution are highlighted in orange. C. 2D peak distribution, plotting the intensities of peaks 2425 Da and 2482 Da across PC3, LNCaP and PNT2 spectra.

Figure 6

PNT2 cells co-cultured with LNCaP cells. A. Mixed population of non-cancerous (PNT2) and cancerous (LNCaP) prostatic cells. Confocal image of LNCaP labelled with CellTracker™_Green (here in red) on a PNT2 cells layer (here in green) cultured for 72h. Blue: Hoechst; Green Phalloidin; Red: LNCaP labelled with CellTracker™_Green. False colors are used for better visibility. Bar : 100 μm. B. 2D peak distribution of MALDI profiling analysis of PNT2 cells co-cultured with LNCaP cells.
PNT2 cells co-cultured with LNCaP cells. A. Mixed population of non-cancerous (PNT2) and cancerous (LNCaP) prostatic cells. Confocal image of LNCaP labelled with CellTracker™_Green (here in red) on a PNT2 cells layer (here in green) cultured for 72h. Blue: Hoechst; Green Phalloidin; Red: LNCaP labelled with CellTracker™_Green. False colors are used for better visibility. Bar : 100 μm. B. 2D peak distribution of MALDI profiling analysis of PNT2 cells co-cultured with LNCaP cells.
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