Research Papers

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

[+] Author and Article Information
Emeline Pagès

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

Murielle Rémy

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

Virginie Kériquel

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

Manuela Medina Correa

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

Bertrand Guillotin

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

Fabien Guillemot

146, rue Léo-Saignat, Case 45,
Bordeaux 33076, France;
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.

Copyright © 2015 by ASME
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Murphy, S. V. , and Atala, A. , 2014, “ 3D Bioprinting of Tissues and Organs,” Nat. Biotechnol., 32(8), pp. 773–785. [CrossRef] [PubMed]
Guillemot, F. , Mironov, V. , and Nakamura, M. , 2010, “ Bioprinting Is Coming of Age: Report From the International Conference on Bioprinting and Biofabrication in Bordeaux (3B’09),” Biofabrication, 2(1), p. 010201. [CrossRef] [PubMed]
Lee, W. , Debasitis, J. C. , Lee, V. K. , Lee, J.-H. , Fischer, K. , Edminster, K. , Park, J.-K. , and Yoo, S.-S. , 2009, “ Multi-Layered Culture of Human Skin Fibroblasts and Keratinocytes Through Three-Dimensional Freeform Fabrication,” Biomaterials, 30(8), pp. 1587–1595. [CrossRef] [PubMed]
Michael, S. , Sorg, H. , Peck, C.-T. , Koch, L. , Deiwick, A. , Chichkov, B. , Vogt, P. M. , and Reimers, K. , 2013, “ Tissue Engineered Skin Substitutes Created by Laser-Assisted Bioprinting Form Skin-Like Structures in the Dorsal Skin Fold Chamber in Mice,” PLoS One, 8(3), p. e57741. [CrossRef] [PubMed]
Koch, L. , Kuhn, S. , Sorg, H. , Gruene, M. , Schlie, S. , Gaebel, R. , Polchow, B. , Reimers, K. , Stoelting, S. , Ma, N. , Vogt, P. M. , Steinhoff, G. , and Chichkov, B. , 2010, “ Laser Printing of Skin Cells and Human Stem Cells,” Tissue Eng., Part C, 16(5), pp. 847–854. [CrossRef]
Gurkan, U. A. , El Assal, R. , Yildiz, S. E. , Sung, Y. , Trachtenberg, A. J. , Kuo, W. P. , and Demirci, U. , 2014, “ Engineering Anisotropic Biomimetic Fibrocartilage Microenvironment by Bioprinting Mesenchymal Stem Cells in Nanoliter Gel Droplets,” Mol. Pharm., 11(7), pp. 2151–2159. [CrossRef] [PubMed]
Cui, X. , Breitenkamp, K. , Finn, M. G. , Lotz, M. , and D'Lima, D. D. , 2012, “ Direct Human Cartilage Repair Using Three-Dimensional Bioprinting Technology,” Tissue Eng., Part A, 18(11–12), pp. 1304–1312. [CrossRef]
Catros, S. , Fricain, J.-C. , Guillotin, B. , Pippenger, B. , Bareille, R. , Remy, M. , Lebraud, E. , Desbat, B. , Amédée, J. , and Guillemot, F. , 2011, “ Laser-Assisted Bioprinting for Creating On-Demand Patterns of Human Osteoprogenitor Cells and Nano-Hydroxyapatite,” Biofabrication, 3(2), p. 025001. [CrossRef] [PubMed]
Jakab, K. , Norotte, C. , Damon, B. , Marga, F. , Neagu, A. , Besch-Williford, C. L. , Kachurin, A. , Church, K. H. , Park, H. , Mironov, V. , Markwald, R. , Vunjak-Novakovic, G. , and Forgacs, G. , 2008, “ Tissue Engineering by Self-Assembly of Cells Printed Into Topologically Defined Structures,” Tissue Eng., Part A, 14(3), pp. 413–421. [CrossRef]
Cui, X. , and Boland, T. , 2009, “ Human Microvasculature Fabrication Using Thermal Inkjet Printing Technology,” Biomaterials, 30(31), pp. 6221–6227. [CrossRef] [PubMed]
Wu, P. K. , and Ringeisen, B. R. , 2010, “ Development of Human Umbilical Vein Endothelial Cell (HUVEC) and Human Umbilical Vein Smooth Muscle Cell (HUVSMC) Branch/Stem Structures on Hydrogel Layers Via Biological Laser Printing (BioLP),” Biofabrication, 2(1), p. 014111. [CrossRef] [PubMed]
Ouyang, L. , Yao, R. , Chen, X. , Na, J. , and Sun, W. , 2015, “ 3D Printing of HEK 293FT Cell-Laden Hydrogel Into Macroporous Constructs With High Cell Viability and Normal Biological Functions,” Biofabrication, 7(1), p. 015010. [CrossRef] [PubMed]
Guillotin, B. , and Guillemot, F. , 2011, “ Cell Patterning Technologies for Organotypic Tissue Fabrication,” Trends Biotechnol., 29(4), pp. 183–190. [CrossRef] [PubMed]
Xu, C. , Zhang, M. , Huang, Y. , Ogale, A. , Fu, J. , and Markwald, R. R. , 2014, “ Study of Droplet Formation Process During Drop-on-Demand Inkjetting of Living Cell-Laden Bioink,” Langmuir, 30(30), pp. 9130–9138. [CrossRef] [PubMed]
Guillotin, B. , Souquet, A. , Catros, S. , Duocastella, M. , Pippenger, B. , Bellance, S. , Bareille, R. , Rémy, M. , Bordenave, L. , Amédée, J. , and Guillemot, F. , 2010, “ Laser Assisted Bioprinting of Engineered Tissue With High Cell Density and Microscale Organization,” Biomaterials, 31(28), pp. 7250–7256. [CrossRef] [PubMed]
Guillemot, F. , Souquet, A. , Catros, S. , Lopez, J. , Faucon, M. , Pippenger, B. , Bareille, R. , Chollet, C. , Rémy, M. , Chabassier, P. , Durrieu, M.-C. , Fricain, J.-C. , and Amédée, J. , 2008, “ High-Throughput Laser Printing of Cells and Biomaterials for Tissue Engineering,” Acta Biomater., 6(7), pp. 2494–2500. [CrossRef]
Guillemot, F. , Souquet, A. , Catros, S. , and Guillotin, B. , 2010, “ Laser-Assisted Cell Printing: Principle, Physical Parameters Versus Cell Fate and Perspectives in Tissue Engineering,” Nanomedicine, 5(3), pp. 507–515. [CrossRef] [PubMed]
Ali, M. , Pages, E. , Ducom, A. , Fontaine, A. , and Guillemot, F. , 2014, “ Controlling Laser-Induced Jet Formation for Bioprinting Mesenchymal Stem Cells With High Viability and High Resolution,” Biofabrication, 6(4), p. 045001. [CrossRef] [PubMed]
Devillard, R. , Pagès, E. , Correa, M. M. , Kériquel, V. , Rémy, M. , Kalisky, J. , Ali, M. , Guillotin, B. , and Guillemot, F. , 2014, “ Cell Patterning by Laser-Assisted Bioprinting,” Methods Cell Biol., 119, pp. 159–174. [PubMed]
Johnson, K. , Hashimoto, S. , Lotz, M. , Pritzker, K. , Goding, J. , and Terkeltaub, R. , 2001, “ Up-Regulated Expression of the Phosphodiesterase Nucleotide Pyrophosphatase Family Member PC-1 Is a Marker and Pathogenic Factor for Knee Meniscal Cartilage Matrix Calcification,” Arthritis Rheum., 44(5), pp. 1071–1081. [CrossRef] [PubMed]
Hsiong, S. X. , Boontheekul, T. , Huebsch, N. , and Mooney, D. J. , 2009, “ Cyclic Arginine-Glycine-Aspartate Peptides Enhance Three-Dimensional Stem Cell Osteogenic Differentiation,” Tissue Eng., Part A, 15(2), pp. 263–272. [CrossRef]
Gruene, M. , Unger, C. , Koch, L. , Deiwick, A. , and Chichkov, B. , 2011, “ Dispensing Pico to Nanolitre of a Natural Hydrogel by Laser-Assisted Bioprinting,” Biomed. Eng. Online, 10, p. 19. [CrossRef] [PubMed]
Eiraku, M. , Takata, N. , Ishibashi, H. , Kawada, M. , Sakakura, E. , Okuda, S. , Sekiguchi, K. , Adachi, T. , and Sasai, Y. , 2011, “ Self-Organizing Optic-Cup Morphogenesis in Three-Dimensional Culture,” Nature, 472(7341), pp. 51–56. [CrossRef] [PubMed]
Lutolf, M. P. , and Hubbell, J. A. , 2005, “ Synthetic Biomaterials as Instructive Extracellular Microenvironments for Morphogenesis in Tissue Engineering,” Nat. Biotechnol., 23(1), pp. 47–55. [CrossRef] [PubMed]


Grahic Jump Location
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).

Grahic Jump Location
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. 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. 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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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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