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Research Papers

Creation of Highly Defined Mesenchymal Stem Cell Patterns in Three Dimensions by Laser-Assisted Bioprinting

[+] Author and Article Information
Emeline Pagès

INSERM U1026,
146, rue Léo-Saignat, Case 45,
Bordeaux 33076, France
e-mail: emeline.pages@inserm.fr

Murielle Rémy

University of Bordeaux;
INSERM U1026,
146, rue Léo-Saignat, Case 45,
Bordeaux 33076, France
e-mail: murielle.remy@u-bordeaux.fr

Virginie Kériquel

INSERM U1026,
146, rue Léo-Saignat, Case 45,
Bordeaux 33076, France
e-mail: virginie.keriquel@orange.fr

Manuela Medina Correa

INSERM U1026,
146, rue Léo-Saignat, Case 45,
Bordeaux 33076, France
e-mail: manuelamedinacorrea@gmail.com

Bertrand Guillotin

INSERM U1026,
146, rue Léo-Saignat, Case 45,
Bordeaux 33076, France
e-mail: gagarstl@free.fr

Fabien Guillemot

INSERM U1026,
146, rue Léo-Saignat, Case 45,
Bordeaux 33076, France;
POIETIS,
Bioparc Bordeaux Métropole,
27 allée Charles Darwin,
Pessac 33600, France
e-mail: fabien.guillemot@poietis.com

Manuscript received May 11, 2015; final manuscript received July 27, 2015; published online September 29, 2015. Assoc. Editor: Ibrahim Ozbolat.

J. Nanotechnol. Eng. Med 6(2), 021006 (Sep 29, 2015) (5 pages) Paper No: NANO-15-1040; doi: 10.1115/1.4031217 History: Received May 11, 2015; Revised July 27, 2015

Bioprinting is a technology that allows making complex tissues from the bottom-up. The need to control accurately both the resolution of the printed droplet and the precision of its positioning was reported. Using a bioink with 1 × 108 cells/mL, we present evidence that the laser-assisted bioprinter (LAB) can deposit droplets of functional mesenchymal stem cells with a resolution of 138 ± 28 μm and a precision of 16 ± 13 μm. We demonstrate that this high printing definition is maintained in three dimensions.

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Figures

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Fig. 6

High-definition cell printing in 3D. A first pattern of D1T cells was printed on a collagen layer (left panel). This pattern was covered with a second collagen layer. By changing the microscope focus while remaining in the same area, another cell pattern was observed (right panel) (and covered again with collagen).

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Fig. 2

Postprinting DNA fragmentation. Four hours after printing, D1 cells were fixed and treated for a TUNEL assay. No fluorescent staining was detected suggesting that there was no DNA fragmentation induced by LAB (left panel). A positive control (nuclease-treated sample, right panel) for the assay is presented. Green fluorescence corresponded to DNA terminal ends.

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Fig. 1

Viability of printed cells. One day after printing, D1 cells adhered to collagen (left panel). The Live/Dead assay suggested that cells were mainly alive (middle panel), any dead cell was detected (right panel).

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Fig. 5

LAB of the cell patterns with high definition. (a) D1T cells, stained by tdTomato, were printed on a collagen layer. The position of each islet of cells varied from 150 μm (top panel) to 500 μm (bottom panel) between two successive islets within a line, with a 50 μm step between each panel. (b) The distance measured between each pair of consecutive islets on the substrate (dm) was calculated using a dedicated python computer program and plotted in function of the distance expected (de).

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Fig. 3

Comparison of printed and unprinted cell proliferation. First, an Alamar Blue assay was performed for different seeding densities. Thus, the fluorescent signal detected was quantified in function of cell density (cells/cm2). Then, an Alamar Blue assay was performed over 4 days. Results for printed (white) and unprinted (black) cells are presented.

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Fig. 4

Ability of cells to differentiate toward osteogenic lineage after the printing process. Printed pluripotent precursors of bone marrow were grown in control medium (control) or in osteoblastic-inductive medium (differentiated). The absorbance measures correspond to the quantity of calcium produced by the cells.

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