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Review
. 2013 Jan;31(1):10-9.
doi: 10.1016/j.tibtech.2012.10.005. Epub 2012 Dec 19.

Bioprinting for stem cell research

Savas Tasoglu  1 , Utkan Demirci
Affiliations
Review

Bioprinting for stem cell research

Savas Tasoglu et al. Trends Biotechnol. 2013 Jan.

Abstract

Recently, there has been growing interest in applying bioprinting techniques to stem cell research. Several bioprinting methods have been developed utilizing acoustics, piezoelectricity, and lasers to deposit living cells onto receiving substrates. Using these technologies, spatially defined gradients of immobilized biomolecules can be engineered to direct stem cell differentiation into multiple subpopulations of different lineages. Stem cells can also be patterned in a high-throughput manner onto flexible implementation patches for tissue regeneration or onto substrates with the goal of accessing encapsulated stem cells of interest for genomic analysis. Here, we review recent achievements with bioprinting technologies in stem cell research, and identify future challenges and potential applications including tissue engineering and regenerative medicine, wound healing, and genomics.

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Figures

Figure 1
Figure 1
Printed heparin-binding epidermal growth factor-like growth factor patterns with uniform, increasing, and decreasing gradients compared to control with no pattern. (a) Image of the cell starting line in contact with the pattern. (b) The growth factor pattern was separated into 4 windows of identical area. The cell count in each window was performed every 24 h. (c) Comparisons of cell responses among control with no pattern, uniform pattern, high-to-low pattern, and low-to-high pattern. Cells were seeded at the origin of the patterns and imaged over time (4 days in culture). 13 overprints were performed for uniform pattern. Lower and upper limit were set to 1 and 25 overprints for increasing and decreasing gradients of patterns. Reproduced with permission[32]. Copyright Year, Publisher.
Figure 2
Figure 2
EB formation after bioprinting method. (a-c) Images of formed EBs with droplet sizes of 1, 4, 10, and 20 μL at a cell density of 105 cells/ ml. (a) Uniform-sized droplets encapsulating ESCs were generated by bioprinting. (b) Phase contrast images of EBs formed after hanging for 24 h and c after culture for 72 h in a 96 multiwell plate. (d) Fluorescent images of GFP positive EBs at t=96 h stained with ethidium homodimer. (e) Images of EBs formed with printed droplet size of 10 μl at t=72 h at different cell concentrations. Reproduced with permission [37]. Copyright Year, Publisher.
Figure 3
Figure 3
Bioprinted patch implementation. (a) Microscopic image of the mesenchymal stem cell (MSC) pattern directly after laser induced forward transfer (LIFT) printing. (b) fluorescence live/dead image of the printed cells after 6 days under chondrogenic culture conditions. (c) Image of MSCs, predifferentiated in osteogenic lineage for 7 days, printed in two chessboard patterns and cultivated for 17 days under osteogenic conditions. (d) Transmission image, (e) fluorescence images of nuclei stained with Hoechst 33342 (blue staining), and (f) membranes stained with DIL (red staining) of the chessboard section. Scale bar is 1 mm. (g) Arrangement of transferred cells by LIFT observed after 24 h: Human MSC were prestained with PKH26 and patches were stained with polyclonal goat anti-Pecam1 24 h after LIFT to separate grid patterned HUVEC. (h) Patch implantation in vivo: After LAD-ligation rats received the cardiac patch sutured onto the area of blanched myocardium. Reproduced with permission. Copyright Year, Publisher.
Figure 4
Figure 4
Cell growth profiles after laser printing on (a) day 0, (b) day 1, (c) day 4, and (d) day 7. EB formation after 7 day culture (scale bar is 500 μm). Fluorescence images of mouse ESCs expressing (e) pluripotency marker, Oct4, and (f) DAPI stain to reveal the cell nuclei. (g) and (h) merged images to show the expression of Oct4 in nuclei (scale bar is 100 μm). The marker proteins of (i and l) nestin (ectoderm), (j and m) Myf-5 (mesoderm) and (k and n) PDX-1 (endoderm), were expressed after 7 days of culture, which confirmed the differentiation potential of mouse ESCs after printing (scale bar is 100 μm for i-k, and 50 μm for l-n). Reproduced with permission[41]. Copyright Year, Publisher.
Figure 5
Figure 5
Comparison of methods for total RNA expression analysis: conventional method vs. a hybrid bioprinting method. (a) Conventional single cell isolation method. 1) Heterogeneous sample was collected from a specific tissue, macro-dissection, 2) cells were stained with a specific antibody, 3) target cells were collected with conventional FACS, 4) multiple dilution steps created a small population of target cells, 5) total RNA gene expression analysis with microarray. (b) Drop-on-demand total RNA analysis utilizing bioprinting method. 1) Heterogeneous sample was collected, 2) cells were stained with antibody and patterned with cell-encapsulating droplets, 3) specific homogeneous samples containing target cells were generated by a cell droplet patterning platform; homogeneous samples were identified by an automated imaging system, 4) total RNA gene expression analysis with microarray. Reproduced with permission [26]. Copyright Year, Publisher.
Box 1, Figure I
Box 1, Figure I
Sketch of bioprinting technologies. (a) Thermal and piezoelectric ink-jet printing. Two major methods to jet the bioink are demonstrated. The thermal technique heats a resistor and expands an air bubble. The piezoelectric technique charges crystals that expand. (b) Setup for acoustic pico-liter droplet generation. Droplets can be deposited drop-on-demand with predetermined separation and locations. Periodically spaced interdigitated gold rings of an acoustic picoliter droplet ejector are demonstrated. The wavelength of the acoustic wave (low ‘f’) is much larger than the cell size resulting in harmless ejection of cells [5]. (c) Sketch of the valve-based printing setup [, –26]. (d) Sketch of the laser printing setup. (left) Laser-guided direct cell printing [18, 39]. The laser is focused into a cell suspension and the force due to difference in refractive indexes moves the cells onto an acceptor substrate (right) The cell-hydrogel compound is propelled forward as a jet by the pressure of a laser-induced vapor bubble.
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