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A novel cell-printing method and its application to hepatogenic differentiation of human adipose stem cell-embedded mesh structures.

Ahn SH, Lee HJ, Lee JS, Yoon H, Chun W, Kim GH - Sci Rep (2015)

Bottom Line: In the shell of the nozzle, a cross-linking agent flowed continuously onto the surface of the dispensed bioink in the core nozzle, so that the bioink struts were rapidly gelled, and any remnant cross-linking solution during the process was rapidly absorbed into the working stage, resulting in high cell-viability in the bioink strut and stable formation of a three-dimensional mesh structure.To demonstrate the applicability of the technique, preosteoblasts and human adipose stem cells (hASCs) were used to obtain cell-laden structures with multi-layer porous mesh structures.The fabricated cell-laden mesh structures exhibited reasonable initial cell viabilities for preosteoblasts (93%) and hASCs (92%), and hepatogenic differentiation of hASC was successfully achieved.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomechatronic Engineering, College of Biotechnology and Bioengineering, Sungkyunkwan University (SKKU), Suwon, South Korea.

ABSTRACT
We report a cell-dispensing technique, using a core-shell nozzle and an absorbent dispensing stage to form cell-embedded struts. In the shell of the nozzle, a cross-linking agent flowed continuously onto the surface of the dispensed bioink in the core nozzle, so that the bioink struts were rapidly gelled, and any remnant cross-linking solution during the process was rapidly absorbed into the working stage, resulting in high cell-viability in the bioink strut and stable formation of a three-dimensional mesh structure. The cell-printing conditions were optimized by manipulating the process conditions to obtain high mechanical stability and high cell viability. The cell density was 1 × 10(7) mL(-1), which was achieved using a 3-wt% solution of alginate in phosphate-buffered saline, a mass fraction of 1.2 wt% of CaCl2 flowing in the shell nozzle with a fixed flow rate of 0.08 mL min(-1), and a translation velocity of the printing nozzle of 10 mm s(-1). To demonstrate the applicability of the technique, preosteoblasts and human adipose stem cells (hASCs) were used to obtain cell-laden structures with multi-layer porous mesh structures. The fabricated cell-laden mesh structures exhibited reasonable initial cell viabilities for preosteoblasts (93%) and hASCs (92%), and hepatogenic differentiation of hASC was successfully achieved.

No MeSH data available.


Optical and fluorescence images of the hASC-laden mesh structures fabricated using the CD-T and CD-CS method and hepatogenic differentiation.(a) Optical images of the mesh structures. (b) Fluorescence images showing live cells in green and dead cells in red after 1 day. (c) DAPI/phalloidin staining after 27 days of culture of the mesh structure. (d) MTT result of the cell-laden structures. Expression levels of hepatocyte-specific genes (e) albumin and (f) TDO-2 using real-time RT-PCR on day 27 (n = 3, mean ± SD, statistical comparison was performed using Student’s t-test, p < 0.01(**)) and (g,h) immunofluorescence images.
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f6: Optical and fluorescence images of the hASC-laden mesh structures fabricated using the CD-T and CD-CS method and hepatogenic differentiation.(a) Optical images of the mesh structures. (b) Fluorescence images showing live cells in green and dead cells in red after 1 day. (c) DAPI/phalloidin staining after 27 days of culture of the mesh structure. (d) MTT result of the cell-laden structures. Expression levels of hepatocyte-specific genes (e) albumin and (f) TDO-2 using real-time RT-PCR on day 27 (n = 3, mean ± SD, statistical comparison was performed using Student’s t-test, p < 0.01(**)) and (g,h) immunofluorescence images.

Mentions: The fabrication conditions and mass fraction of alginate were the same as those used to fabricate the preosteoblast-laden mesh structure shown in Fig. 5. The cell density of hASCs in the mesh structure was 1 × 107 mL−1. Figure 6(a) shows an optical microscope image of the cell-laden structures, revealing a highly porous structure, with dimensions of 10 × 10 × 3.6 mm3. Figure 6(b) shows fluorescence images, showing that the cell viability after 1 day in the porous structure fabricated using CD-CS was 92.3 ± 3%, while the structure using CD-T was 89.6%. Figure 6(c) shows DAPI/Phallodin images after 27 days and those demonstrate the hASCs were more widely distributed on the structure fabricated by CD-CS than that of CD-T process. Figure 6(d) shows the proliferation of viable hASCs, which demonstrate that the cells in the structure fabricated by CD-CS were well alive and exhibited metabolic function during the culture period compared with the structure fabricated by CD-T.


A novel cell-printing method and its application to hepatogenic differentiation of human adipose stem cell-embedded mesh structures.

Ahn SH, Lee HJ, Lee JS, Yoon H, Chun W, Kim GH - Sci Rep (2015)

Optical and fluorescence images of the hASC-laden mesh structures fabricated using the CD-T and CD-CS method and hepatogenic differentiation.(a) Optical images of the mesh structures. (b) Fluorescence images showing live cells in green and dead cells in red after 1 day. (c) DAPI/phalloidin staining after 27 days of culture of the mesh structure. (d) MTT result of the cell-laden structures. Expression levels of hepatocyte-specific genes (e) albumin and (f) TDO-2 using real-time RT-PCR on day 27 (n = 3, mean ± SD, statistical comparison was performed using Student’s t-test, p < 0.01(**)) and (g,h) immunofluorescence images.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4544022&req=5

f6: Optical and fluorescence images of the hASC-laden mesh structures fabricated using the CD-T and CD-CS method and hepatogenic differentiation.(a) Optical images of the mesh structures. (b) Fluorescence images showing live cells in green and dead cells in red after 1 day. (c) DAPI/phalloidin staining after 27 days of culture of the mesh structure. (d) MTT result of the cell-laden structures. Expression levels of hepatocyte-specific genes (e) albumin and (f) TDO-2 using real-time RT-PCR on day 27 (n = 3, mean ± SD, statistical comparison was performed using Student’s t-test, p < 0.01(**)) and (g,h) immunofluorescence images.
Mentions: The fabrication conditions and mass fraction of alginate were the same as those used to fabricate the preosteoblast-laden mesh structure shown in Fig. 5. The cell density of hASCs in the mesh structure was 1 × 107 mL−1. Figure 6(a) shows an optical microscope image of the cell-laden structures, revealing a highly porous structure, with dimensions of 10 × 10 × 3.6 mm3. Figure 6(b) shows fluorescence images, showing that the cell viability after 1 day in the porous structure fabricated using CD-CS was 92.3 ± 3%, while the structure using CD-T was 89.6%. Figure 6(c) shows DAPI/Phallodin images after 27 days and those demonstrate the hASCs were more widely distributed on the structure fabricated by CD-CS than that of CD-T process. Figure 6(d) shows the proliferation of viable hASCs, which demonstrate that the cells in the structure fabricated by CD-CS were well alive and exhibited metabolic function during the culture period compared with the structure fabricated by CD-T.

Bottom Line: In the shell of the nozzle, a cross-linking agent flowed continuously onto the surface of the dispensed bioink in the core nozzle, so that the bioink struts were rapidly gelled, and any remnant cross-linking solution during the process was rapidly absorbed into the working stage, resulting in high cell-viability in the bioink strut and stable formation of a three-dimensional mesh structure.To demonstrate the applicability of the technique, preosteoblasts and human adipose stem cells (hASCs) were used to obtain cell-laden structures with multi-layer porous mesh structures.The fabricated cell-laden mesh structures exhibited reasonable initial cell viabilities for preosteoblasts (93%) and hASCs (92%), and hepatogenic differentiation of hASC was successfully achieved.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomechatronic Engineering, College of Biotechnology and Bioengineering, Sungkyunkwan University (SKKU), Suwon, South Korea.

ABSTRACT
We report a cell-dispensing technique, using a core-shell nozzle and an absorbent dispensing stage to form cell-embedded struts. In the shell of the nozzle, a cross-linking agent flowed continuously onto the surface of the dispensed bioink in the core nozzle, so that the bioink struts were rapidly gelled, and any remnant cross-linking solution during the process was rapidly absorbed into the working stage, resulting in high cell-viability in the bioink strut and stable formation of a three-dimensional mesh structure. The cell-printing conditions were optimized by manipulating the process conditions to obtain high mechanical stability and high cell viability. The cell density was 1 × 10(7) mL(-1), which was achieved using a 3-wt% solution of alginate in phosphate-buffered saline, a mass fraction of 1.2 wt% of CaCl2 flowing in the shell nozzle with a fixed flow rate of 0.08 mL min(-1), and a translation velocity of the printing nozzle of 10 mm s(-1). To demonstrate the applicability of the technique, preosteoblasts and human adipose stem cells (hASCs) were used to obtain cell-laden structures with multi-layer porous mesh structures. The fabricated cell-laden mesh structures exhibited reasonable initial cell viabilities for preosteoblasts (93%) and hASCs (92%), and hepatogenic differentiation of hASC was successfully achieved.

No MeSH data available.