Abstract
Tissue engineering is an essential component of developing effective regenerative therapies. In this study, we introduce a promising method to create scaffold-free three-dimensional (3D) tissue engineered multilayered microstructures from cultured cells using the “3D tissue fabrication system” (Regenova®; Cyfuse, Tokyo, Japan). This technique utilizes the adhesive nature of cells. When cells are cultured in nonadhesive wells, they tend to aggregate and form a spheroidal structure. The advantage of this approach is that cellular components can be mixed into one spheroid, thereby promoting the formation of extracellular matrices, such as collagen and elastin. This system enables one to create a predesigned 3D structure composed of cultured cells. We found that the advantages of this system to be (1) the length, size, and shape of the structure that were designable and highly reproducible because of the computer controlled robotics system, (2) the graftable structure could be created within a reasonable period (8 days), and (3) the constructed tissue did not contain any foreign material, which may avoid the potential issues of contamination, biotoxicity, and allergy. The utilization of this robotic system enabled the creation of a 3D multilayered microstructure made of cell-based spheres with a satisfactory mechanical properties and abundant extracellular matrix during a short period of time. These results suggest that this new technology will represent a promising, attractive, and practical strategy in the field of tissue engineering.
Impact statement
The utilization of the “three dimensional tissue fabrication system” enabled the creation of a three-dimensional (3D) multilayered microstructure made of cell-based spheres with a satisfactory mechanical properties and abundant extracellular matrix during a short period of time. These results suggest that this new technology will represent a promising, attractive, and practical strategy in the field of tissue engineering.
Keywords: tissue engineering, scaffold free, three-dimensional, biofabrication, extracellular matrix
Introduction
Tissue engineering is an essential component of developing effective regenerative therapies.1 With the traditional tissue engineering paradigm of cells, signals, and scaffolds, the field of biomedical engineering has made great progress toward addressing clinical needs. The vast majority of tissue engineering research has relied on the use of scaffolds, and several products are now commercially available. Scaffold-based tissue engineering is particularly popular and includes technologies such as biodegradable scaffolds,2 decellularized tissues,3 hydrogel and cell mixtures,4,5 bioprinting,6 and fiber-based tissue engineering.7
On the contrary, we have focused on a scaffold-free tissue engineering-based approach and have utilized cell sheet technology.8,9 Currently, cell sheet fabrication is tedious and time-consuming leading to low-throughput with variability in the end product.10,11 Cell sheet fabrication is unable to organize itself into complex 3D structures.9–12 In the current study, we have used the “three-dimensional (3D) tissue fabrication system” (Regenova®; Cyfuse, Tokyo, Japan), which is a promising robotic platform equipped with a precise image recognition system for spheroid and needle location to create 3D multicomponent. We hypothesized that this system will enable the creation of a scaffold free, 3D tissue engineered structure composed of cell-based spheres in a highly reproducible manner. We also hypothesized that this 3D multilayered microstructure would provide satisfactory mechanical properties and abundant extracellular matrix (ECM) during a short period of time. The aim of this study was to describe the methodology, examine the biological features, and elasticity of the 3D microstructures consisting of cultured cells.
Materials and Methods
Culture of adipose tissue-derived stem cells, and cell-spheroids preparation
StemPro™ human adipose-derived stem cells (ADSC) were purchased (ThermoFisher Scientific, Inc., Waltham, MA) and cultured in a medium with Corning® stemgro® hMSC Medium (Mediatech, Inc., Manassas, VA) on noncoated culture dishes at 37°C and 5% CO2. The cells were passaged every 2 days and were used within two to five passages.
After incubation, the adherent cells were washed and collected, then plated onto each well of ultralow attachment round-shaped 96-U-well plates PrimeSurface® (Sumitomo Bakelite Co. Ltd., Tokyo, Japan) filled with a culture medium. After 48 h, the cells aggregated to form a round-shaped ADSC spheroid. The size of the ADSC spheroid was measured.
Robotic system to create 3D multilayered microstructures using needle array bioprinting system
We used the Regenova to assemble ADSC spheroids for constructing scaffold-free multilayered microstructures. According to a 3D structure predesigned on a computer system, the Regenova skewers ADSC spheroids into a 9 × 9 needle array. The outer diameter of each needle was 0.17 mm and the distance between each needle was 0.4 mm. The size of the needle array was a square, 3.2 mm in length on each side.
In this system, the ADSCs were aspirated by a robotically controlled fine suction nozzle (O.D. of 0.45 mm and I.D. of 0.23 mm) from the 96-well plate and inserted into the needle-array made of multiple medical-grade stainless needles. A total of 500 ADSC spheroids were created into a 3D structure robotically according to the predesigned configuration. The time required for the placement was ∼1.3 h. Ten days after the placement of the ADSC spheroids on to the needle-array, the needle-array was removed.
Characterization of 3D multilayered microstructures created by Regenova
Samples were visually characterized with scanning electron microscopy (SEM) to confirm the following: (1) cells were densely adherent without an artificial scaffold in a 3D tissue engineered multilayered tissue microstructures, (2) ECM was deposited on the basal surface of a 3D tissue engineered multilayered tissue microstructures, and (3) a 3D tissue engineered multilayered tissue microstructures was preserved. Samples for SEM were fixed for 24 h at 4°C with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 mol/L Sodium Cacodylate Buffer (pH 7.2). Samples were rinsed in the same buffer and postfixed for 1 h with 1% aqueous OsO4. After dehydration in an ascending ethanol series (50%, 70%, 90%, 100% [2 × ]; 10 min each), samples were transferred to 70 micron microporous capsules (Electron Microscopy Sciences, Hatfield, OA) and critical point dried with liquid CO2 in a Tousimis Autosamdri-815B apparatus (Tousimis, Rockville, MD). Microspheres were mounted with conductive copper tape onto 15 mm aluminum stubs (Electron Microscopy Sciences, Hatfield, PA) and sputter coated with 50 Å of gold-palladium using a Denton Desk II Sputter Coater (Denton Vacuum, Moorestown, NJ). Visualization was performed with a Zeiss Sigma FESEM (Carl Zeiss Microscopy, Jena, Germany) operated at 2–3 kV, using in Lens SE detection at a working distance of 5–6 mm. Images were captured in TIFF format using a store resolution of 2048 × 1536 and a line averaging, noise reduction algorithm.13
Mechanical testing of 3D multi-layered microstructures created by 3D tissue fabrication system
Mechanical properties of 3D tissue engineered microstructures created by Regenova were evaluated. The ring-shaped 3D tissue engineered microstructure was prepared by Regenova (n = 3). Uniaxial tensile testing was conducted with a Tissue puller 560TP (Danish myo technology, Aarhus, DNK). The specimens were mounted through two hooks. Upper hook pulled the specimens and the tensile strength was measured. Target velocity of the hook was set at 50 μm/s. A stress-strain phase at incremental displacements of 0.5 mm were performed, followed by a ramp to failure at 0.1 mm/s (Supplementary Video S1). A custom image acquisition software system (Digi-Velpo, version 1.4.1, run in LabView 2010; National Instruments, Austin, TX) was used to acquire optical data on tissue displacement. Optical strain data were integrated with the Instron load data using Opticus (custom-made software run in MATLAB, Natick, MA), which generated stress-strain curves for each sample.
Histological examination of cultured ADSC and the 3D multilayered microstructures created by Regenova
The tissue samples were fixed in 4% paraformaldehyde, and embedded in paraffin for 5 mm thick sections. The paraffin-embedded sections were used for routine hematoxylin–eosin (HE) staining to assess the structure of tissue engineered material. Elastica-Masson was performed to assess elastic microfiber networks in the tissue engineered materials. To determine the extent of apoptosis, tissue samples were subjected to terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) with an in situ Apoptosis Detection kit (MK 500; Takara Bio, Inc., Shiga, Japan).
The tissue sections were deparaffined with xylene, and rehydrate through an ethanol series and PBS. Antigen retrieval was performed by Proteinase K treatment. Endogenous peroxidase was blocked with 0.3% H2O2 in methanol for 30 min, followed by incubation with Protein Block (Genostaff, Tokyo, Japan) and Avidin/Biotin Blocking Kit (Vector Laboratories, Inc., Burlingame, CA). To detect collagen accumulation, the sections were incubated with anticollagen I (COL I) mouse monoclonal antibody (AB90395, 1 μg/mL; Abcam, Cambridge, United Kingdom) or anticollagen II (COL II) mouse monoclonal antibody (NBP2-33343, 5 μg/mL; NOVUS Biologicals, Littleton, CO) at 4°C overnight. They were incubated with biotin-conjugated goat anti-mouse Ig (BA-9200; Vector Laboratories, Inc.) for 30 min at room temperature followed by the addition of peroxidase conjugated streptavidin (Nichirei Biosciences, Inc., Tokyo, Japan) for 5 min. Peroxidase activity was visualized by diaminobenzidine. The sections were counterstained with Mayer's Hematoxylin (MUTO, Tokyo, Japan), dehydrated, and then mounted with Malinol (MUTO). The images were examined by microscopy (DM 2500; Leica, Wetzlar, Germany).
Statistical analysis
Continuous variables are expressed as the median and interquartile range (IQR). Comparisons between two groups were made with the Wilcoxon–Mann–Whitney U test. For comparisons between three groups, we used the Kruskal–Wallis test followed by post hoc pairwise Wilcoxon–Mann–Whitney U tests. The multiplicity in pairwise comparisons was corrected by the Bonferroni procedure. For categorical variables, two groups were compared with the Fisher exact test. A p-value <0.05 was considered statistically significant. All calculations were performed with JMP 9.0 (SAS Institute, Inc., Cary, NC).14
Results
Culture of adipose tissue-derived stem cells and cell-spheroids preparation
StemPro human ADSC were purchased (ThermoFisher Scientific, Inc.) and cultured in a medium with Corning stemgro hMSC Medium (Mediatech, Inc.) on noncoated culture dishes at 37°C and 5% CO2. The cells were passaged every 2 days and were used within the two to five passages (Fig. 1 A).
FIG. 1.
The robotic system to create 3D multilayered tissue engineered microstructures using needle array bioprinting system. (A) StemPro™ human ADSC (ThermoFisher Scientific, Inc., Waltham, MA) were cultured in a medium with Corning® stemgro® hMSC Medium (Mediatech, Inc., Manassas, VA) on noncoated culture dishes at 37°C and 5% CO2. Bar = 200 μm. (B) After incubation, the adherent cells were plated onto each well of ultralow attachment round-shaped 96-U-well plates PrimeSurface® (Sumitomo Bakelite Co. Ltd.) filled with a culture medium. After 48 h, the cells aggregated to form a round-shaped ADSC spheroid. The size of the ADSC spheroid was median of 525 μm (n = 4, IQR 506–544 μm). Bar = 200 μm. (C, D) “3D tissue fabrication system” (Regenova®; Cyfuse, Tokyo, Japan) was used to assemble ADSC spheroids for constructing scaffold-free multilayered tissue engineered microstructures. According to a 3D structure predesigned on a computer system, the Regenova skewers ADSC spheroids into a 9 × 9 needle array in a ring shape. (C, D) Showed the upper and side views of the tissue engineered microstructure immediately after the placement of the ADSC spheroids, respectively. Bar = 2 mm. (E–G) Six days after the placement of the ADSC spheroids on to the needle array, the needle array was removed. (E, F) Showed the upper and side views of structure 6 days after the placement of the ADSC spheroids, respectively. (G) Showed the tissue engineered microstructure after the needle array was removed. Bar = 2 mm. (H, I) Ten days after the placement of the ADSC spheroids. The configuration of the structure was slightly shrunk, but retained after the removal from the needle array. Bar = 2 mm. ADSC, adipose-derived stem cells; 3D, three-dimensional; IQR, interquartile range.
After incubation, the adherent cells were washed and collected, then plated onto each well of ultralow attachment round-shaped 96-U-well plates PrimeSurface (Sumitomo Bakelite Co. Ltd.) filled with a culture medium. After 48 h, the cells aggregated to form a round-shaped ADSC spheroid. The size of the ADSC spheroid was median of 525 μm (n = 4, IQR 506–544 μm) (Fig. 1B).
Robotic platform to create 3D multilayered microstructures
We used the Regenova to assemble ADSC spheroids for constructing scaffold-free multilayered microstructures. Three-dimensional structures were predesigned on a computer system, the Regenova skewers ADSC spheroids into a 9 × 9 needle array (Fig. 1C, D). The outer diameter of each needle was 0.17 mm and the distance between each needle was 0.4 mm. The size of the needle array was a square, 3.2 mm in length on each side. In this system, the ADSCs were aspirated by a robotically controlled fine suction nozzle (O.D. of 0.45 mm and I.D. of 0.23 mm) from the 96-well plate and inserted into the needle array made of multiple medical grade stainless needles. A total of 500 ADSC spheroids were robotically grafted into a 3D structure according to the predesigned configuration (Fig. 1E–G). The time required for the placement was ∼1.3 h. Ten days after the placement of the ADSC spheroids on to the needle array, the needle array was removed. Over this 10 day period, there was a small decrease in diameter of the configuration, but after the removal from the needle array, it retained its dimension due to fusion between the ADSC (Fig. 1H, I).
Geometrical characterization of 3D multilayered microstructures created by 3D tissue fabrication system
Samples were visually characterized with SEM to confirm the following: (1) cells were densely adherent without an artificial scaffold in a 3D tissue engineered multilayered tissue microstructures, (2) ECM was deposited on the basal surface of a 3D tissue engineered multilayered tissue microstructures, and (3) a 3D tissue engineered multilayered tissue microstructures was preserved. Samples for SEM were fixed for 24 h at 4°C with 4% paraformaldehyde and 2% glutaraldehyde in 0.1 mol/L Sodium Cacodylate Buffer (pH 7.2). Samples were rinsed in the same buffer and postfixed for 1 h with 1% aqueous OsO4. After dehydration in an ascending ethanol series (50%, 70%, 90%, 100% [2 × ]; 10 min each), samples were transferred to 70 micron microporous capsules (Electron Microscopy Sciences, Hatfield, OA) and critical point dried with liquid CO2 in a Tousimis Autosamdri-815B apparatus (Tousimis). Microspheres were mounted with conductive copper tape onto 15 mm aluminum stubs (Electron Microscopy Sciences, Hatfield, PA) and sputter coated with 50 Å of gold-palladium using a Denton Desk II Sputter Coater (Denton Vacuum). Visualization was performed with a Zeiss Sigma FESEM (Carl Zeiss Microscopy) operated at 2–3 kV, using in Lens SE detection at a working distance of 5–6 mm. Images were captured in TIFF format using a store resolution of 2048 × 1536 and a line averaging, noise reduction algorithm.13
Using high-resolution SEM to characterize multilayered ADSC microspheres, various ultrastructural features became evident. Spheres were strongly connected to form an integrated 3D structure, with regular holes where the needle array of the Regenova was removed (Fig. 2A). The confluent and integrated cell layers (Fig. 2B) as well as abundance of collagen (Fig. 2C, D) were also suggested by mechanical testing, confirming the relationship between stress and strain. The microstructure was unperturbed by the removal of needles, and pores showed a lining of closely adherent cells and collagen (Fig. 2D). The smooth cell surface versus fibrous collagen layer can be seen (Fig. 2D).
FIG. 2.
Characterization of 3D multilayered tissue engineered microstructures using high resolution SEM. (A) Spheres were strongly connected to form an integrated 3D structure, with regular holes where the needle array from the needle array of the Regenova was removed. (B) The confluent and integrated cell layers (white arrow). (C) Abundance of collagen (white arrow). (D) Microstructure seems unperturbed by the removal of needles, and pores similarly show a lining of closely adherent cells and collagen (short white arrow and yellow asterisks). The smooth cell surface versus fibrous collagen layer can be seen. SEM, scanning electron microscopy.
Mechanical characterization of 3D multilayered microstructures created by 3D tissue fabrication system
Mechanical properties of 3D tissue engineered microstructures created by Regenova were evaluated. The ring-shaped 3D tissue engineered microstructure was prepared by Regenova (n = 3). Uniaxial tensile testing was conducted with a Tissue puller 560TP (Danish myo technology). The specimens were mounted through two hooks. The upper hook pulled the specimens and the tensile strength was measured. Target velocity of the hook was set at 50 μm/s. A stress-strain phase at incremental displacements of 0.5 mm was performed, followed by a ramp to failure at 0.1 mm/s (Supplementary Video S1). A custom image acquisition software system (Digi-Velpo, version 1.4.1, run in LabView 2010; National Instruments) was used to acquire optical data on tissue displacement.12 Optical strain data were integrated with the Instron load data using Opticus (custom-made software run in MATLAB, Natick, MA), which generated stress-strain curves for each sample (Fig. 3). The elastic modulus, which was calculated from the slope of the stress-strain curves, was median of 0.177 MPa (IQR 0.135–0.221 MPa).
FIG. 3.
Mechanical properties of 3D tissue engineered microstructures created by Regenova were evaluated. Uniaxial tensile testing was conducted with an Instron 5543 Microtester (Instron, Norwood, Mass). Optical strain data were integrated with the Instron load data using Opticus (custom-made software run in MATLAB, Natick, Mass), which generated stress-strain curves for each ventricular strip. The elastic modulus, which was calculated from the slope of the stress-strain curves, was median of 0.177 MPa (n = 3, IQR 0.135–0.221 MPa).
Histological characterization of cultured ADSC and the 3D multilayered microstructures created by 3D tissue fabrication system
The tissue samples were fixed in 4% paraformaldehyde, and embedded in paraffin for 5 mm thick sections. The paraffin-embedded sections were used for routine HE staining to assess the structure of tissue engineered material. Elastica-Masson was performed to assess elastic microfiber networks in the tissue engineered materials. To evaluate the extent of apoptosis, a form of programmed cell death, tissue samples were subjected to TUNEL with an in situ Apoptosis Detection kit (MK 500; Takara Bio, Inc.).
The tissue sections were deparaffined with xylene and rehydrated through an ethanol series and PBS. Antigen retrieval was performed by Proteinase K treatment. Endogenous peroxidase was blocked with 0.3% H2O2 in methanol for 30 min, followed by incubation with Protein Block (Genostaff) and Avidin/Biotin Blocking Kit (Vector Laboratories, Inc.). To detect collagen accumulation, the sections were incubated with anticollagen I (COL I) mouse monoclonal antibody (AB90395, 1 μg/mL; Abcam) or anticollagen II (COL II) mouse monoclonal antibody (NBP2-33343, 5 μg/mL; NOVUS Biologicals) at 4°C overnight. They were incubated with biotin-conjugated goat anti-mouse Ig (BA-9200; Vector Laboratories, Inc.) for 30 min at room temperature followed by the addition of peroxidase conjugated streptavidin (Nichirei Biosciences, Inc.) for 5 min. Peroxidase activity was visualized by diaminobenzidine. The sections were counterstained with Mayer's Hematoxylin (MUTO), dehydrated, and then mounted with Malinol (MUTO).
The layer-by-layer structure was well preserved, as assessed by HE staining (Fig. 4A, B). The elastic fiber content was abundant within the sample, as shown by Elastica-Masson staining (Fig. 4C, D). Immunostaining images showed abundant anticollagen I-positive cells (Fig. 4E, F), but a small amount of anticollagen II-positive cells within the sample (Fig. 4G, H). Only a small number of TUNEL-positive cells were found within the sample (Fig. 4I, J).
FIG. 4.
(A, B) Representative macroscopic (A, × 16) and microscopic (B, × 100) HE staining of the sample. The layer-by-layer structure was well preserved. (C, D) Representative macroscopic (C, × 16) and microscopic (D, × 100) Elastica-Masson staining of the sample. The elastic fiber content was abundant within the sample. (E, F) Immunostaining macroscopic (E, × 16) and microscopic (F, × 100) images showed abundant anticollagen I-positive cells within the sample. (G, H) Immunostaining macroscopic (G, × 16) and microscopic (H, × 100) images showed a small amount of anticollagen II-positive cells within the sample. (I, J) Immunostaining macroscopic (I, × 16) and microscopic (J, × 100) images showed a small amount of TUNEL-positive cells within the sample. HE, hematoxylin–eosin; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.
Discussion
We developed a promising method to create scaffold-free 3D tissue engineered multilayered microstructures from cultured ADSC spheres using the “3D tissue fabrication system.” In this study, we described geometrical, mechanical, and histological characterization of this 3D multilayered microstructures created by this 3D tissue fabrication system. This technique utilized the adhesive nature of the cells. When the cells were cultured in nonadhesive wells, they tended to aggregate with each other and form a spheroidal structure within 24 h. The advantage of this approach was that cellular components can be mixed into one spheroid, thereby promoting the formation of extracellular matrices, such as collagen and elastin. This system enables the creation of a predesigned 3D structure that cultured cells can be transferred to. The advantages of this system were as follows: (1) the length, size, and shape of the structure were designable and highly reproducible because of the computer controlled robotics system, (2) the graftable structure could be created within a reasonable period (8 days), and (3) the constructed tissue did not contain any foreign material, which may avoid the potential issues such as contamination, biotoxicity, and allergy.
To date, we have developed a scaffold-free cell sheet technology.9–12 This method has shown a significant potential in that no animal-derived or foreign substances were used. The practical problem of this approach, however, was the handling characteristics at the time of delivery. In this study, we created a 3D tissue engineered structure during a short period of time with a superior tensile property. To our knowledge, this is the first report to describe the mechanical properties of this scaffold-free 3D tissue engineered multilayered microstructures. The elastic modulus assessed from the slope of the stress-strain curves demonstrated 0.177 MPa. Just as a reference, it has been reported that the elastic modulus of canine saphenous vein and ascending aorta showed 0.03 and 0.48 MPa,15 respectively. These data suggest that the 3D tissue engineered material can be stiffer than in vivo vein graft and more flexible than in vivo arterial graft, although this is not a direct comparison. Since the mechanical properties of this tissue engineered graft are one of the most fundamental performance criteria for clinical usage, graft compliance, as examined here by the ultimate tensile strengths and burst pressure of these microstructures, is considered sufficient to retain integrity and resist permanent deformation. Graft compliance and the way in which deformation under loading occurs will also be important as adverse biological responses, which may be associated with compliance mismatch between native tissue and both synthetic and biological tissue engineered grafts. Although the mechanism for this tensile profile was not clear, it was apparent that the 3D tissue material was geometrically coated with an abundant ECM, rich with collagen and elastin components, as shown by our histological observation of elastin and collagen fibers in the construct. The elastic fiber network has been well known to contribute to the essential properties of elasticity and recoil, along with maintaining the integrity of tissue architecture.16 Moreover, the ECM may contribute to the reorganization of the ADSC within the microstructures after the ADSC fuse to each other. Furthermore, the refinement of this structural fabrication process may be evoked to generate elastic fibers in the biostructure. Future interest may be on the impact of additional elastin and collagen over the mechanical properties of this tissue engineered microstructure, as well as how these may change over longer periods of in vitro cell culture or in vivo. Our preliminary data suggest that the additional growth factors can make microstructures stiffer, which seems to be associated with overexpression of collagen I (data not shown). Looking forward, the effect of the content of culture medium and the duration of culturing cells may be of interest for further study.
Although we applied ADSC in this study, several different cell types can potentially be used in the in vitro and in vivo preparation of this scaffold-free 3D tissue engineered multilayered microstructures. The type of cells used can potentially affect the structure of the graft and ultimately how it performs in vivo, along with impacting the graft manufacturing process. Itoh et al. reported the multicellular spheroids based tubular tissues underwent remodeling and endothelialization following implanted in rat aorta.17 Regarding ADSC as a cell source, previous reports, including ours, showed that human adipose tissue-derived stem cells were able to differentiate into mature endothelial cells, contribute to blood vessel formation, thereby improving the survival and organization of implanted cells by maintaining a minimum intercapillary distance to provide oxygen and nutrients.9,10
This method of tissue engineering took advantage of the capacity of dissociated cells to aggregate through cell–cell attachment. Although it is possible to create a multilayered cell sheet, at some point there will be compromised cellular survival due to lack of efficient energy source leading to cell death. In this study, our histological observation showed only a minimal amount of apoptosis within the created microstructure. These results suggest that the cell–cell attachment phenomenon could prevent cell death, possibly by activating signals mediated by ECM receptors and ligands which suppress the anoikis cascade. This phenomenon has been shown to occur in almost all living organisms irrespective of their complexity.18
From the technical aspect, it is possible to control the total number of cells and the type of cells. In the current study, the total number of ADSC was set to 1.0 × 104 cells per each sphere. This number was set due to the relationship between the size of the tip of the robotically controlled nozzle (400 μm) and the created sphere diameter. Therefore, this 3D tissue fabrication system is extremely precise for controlling the length, size, and shape of the structure, and holds great potential for cell studies in the future. It may also be beneficial to examine the effect of the total number of cells on the mechanical characterization of provided materials. The regular holes from the needle array of the Regenova, which were not visible neither macroscopically nor microscopically but visible on SEM, are supposed to be closely related with cell proliferation, however, it needs further investigation to assess the impact of these holes.
Conclusion
The utilization of this robotic platform using a needle array bioprinting system enables the creation of a 3D multilayered microstructure made of cell-based spheres with an excellent mechanical properties and abundant ECM during a short period of time. These results suggest that this new technology will represent a promising, attractive, and practical strategy in the field of tissue engineering.
Supplementary Material
Authors' Contribtions
Y.S., J.W.M., and Y.J.W. wrote the main article text; Y.K. and J.O. prepared Figures 1, 3, and 4; L.J., K.J.J., and A.E. prepared Figure 2. All authors reviewed the article.
Disclosure Statement
No competing financial interests exist.
Funding Information
This study was supported by the National Institutes of Health (NIH) Grant 1R01HL089315-01 (Y.J.W): ISHLT/O.H. Frazier Award in MCS Translational Research, The International Society of Heart & Lung Transplantation (Y.S.).
Supplementary Material
References
- 1. Langer R., and Vacanti J.P.. Tissue engineering. Science 260, 920, 1993 [DOI] [PubMed] [Google Scholar]
- 2. Atluri P., Trubelja A., Fairman A.S., et al. Normalization of post-infarct biomechanics utilizing a novel tissue engineered angiogenic construct. Circulation 128 (26 Suppl 1), S95, 2013 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Neumann A., Sarikouch S., Breymann T., et al. Early systemic cellular immune response in chindren and young adults receiving decellularized fresh allografts for pulmonary valve replacement. Tissue Eng Part A 20, 1003, 2014 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Cohen J.E., Purcell B.P., MacArthur J.W. Jr, et al. A bioengineered hydrogel system enables targeted and sustained intramyocardial delivery of neuregulin, activating the cardiomyocyte cell cycle and enhancing ventricular function in a murine model of ischemic cardiomyopathy. Circ Heart Fail 7, 619, 2014 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. MacArthur J.W., Purcell B., Shudo Y., et al. Sustained release of engineered stromal cell-derived factor 1-alpha from injectable hydrogels effectively recruits endothelial progenitor cells and preserves ventricular function following myocardial infarction. Circulation 128 (26 Suppl 1), S79, 2013 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Murphy S.V., and Atala A.. 3D bioprinting of tissues and organs. Nat Biotechnol 32, 773, 2014 [DOI] [PubMed] [Google Scholar]
- 7. Onoe H., Okitsu T., Itoh A., et al. Metre-long cell-laden microfibers exhibit tissue morphologies and functions. Nat Mater 12, 584, 2013 [DOI] [PubMed] [Google Scholar]
- 8. Okano T., Yamada N., Sakai H., and Sakurai Y.. A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly (N-iso-propylacrylamide). J Biomed Mater Res 27, 1243, 1993 [DOI] [PubMed] [Google Scholar]
- 9. Shudo Y., Miyagawa S., Ohkura H., et al. Addition of mesenchymal stem cells enhances the therapeutic effects of skeletal myoblast cell-sheet transplantation in a rat ischemic cardiomyopathy model. Tissue Eng Part A 20, 728, 2014 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Shudo Y., Miyagawa S., Ohkura H., et al. Adipose tissue-derived multi-lineage progenitor cells improve left ventricular dysfunction in porcine ischemic cardiomyopathy model. J Heart Lung Transplant 36, 237, 2017 [DOI] [PubMed] [Google Scholar]
- 11. Shudo Y., Cohen J.E., MacArthur J.W., et al. Spatially-oriented, temporally-sequential SMC-EPC bi-level cell-sheet neovascularizes ischemic myocardium. Circulation 128 (26 Suppl 1), S59, 2013 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Shudo Y., Cohen J.E., MacArthur J.W., et al. A tissue-engineered chondrocyte cell sheet induces extracellular matrix modification to enhance ventricular biomechanics and attenuate myocardial stiffness in ischemic cardiomyopathy. Tissue Eng Part A 21, 2515, 2015 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Shudo Y., Cohen J.E., Goldstone A.B., et al. Isolation and trans-differentiation of mesenchymal stromal cells into smooth muscle cells: utility and applicability for cell-sheet engineering. Cytotherapy 18, 510, 2016 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Shudo Y., Goldstone A.B., Cohen J.E., et al. Layered smooth muacle cell-endothelial progenitor cell sheets derived from the bone marrow augment postinfarction ventricular function. J Thorac Cardiovasc Surg 154, 955, 2017 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Wesly R.L., Vaishnav R.N., Fuchs J.C., Patel D.J., and Greenfield J.C.. Jr. Static linear and nonlinear elastic properties of normal and arterialized venous tissue in dog and man. Circ Res 37, 509, 1975 [DOI] [PubMed] [Google Scholar]
- 16. Nakamura T., Lozano P.R., Ikeda Y., et al. Fibulin-5/DANCE is essential for elastogenesis in vivo. Nature 415, 171, 2002 [DOI] [PubMed] [Google Scholar]
- 17. Itoh M., Nakayama K., Noguchi R., et al. Scaffold-free tubular tissues created by a Bio-3D printer undergo remodeling and endotholialization when implanted in rat aorta. PlosOne 10, e0136681, 2015 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. Zvibel I., Smets F., and Soriano H.. Anoikis: roadblock to cell transplantation? Cell Transplant 11, 621, 2002 [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.