Microtissues Enhance Smooth Muscle Differentiation and Cell Viability of hADSCs for Three Dimensional Bioprinting

Abstract

Smooth muscle differentiated human adipose derived stem cells (hADSCs) provide a crucial stem cell source for urinary tissue engineering, but the induction of hADSCs for smooth muscle differentiation still has several issues to overcome, including a relatively long induction time and equipment dependence, which limits access to abundant stem cells within a short period of time for further application. Three-dimensional (3D) bioprinting holds great promise in regenerative medicine due to its controllable construction of a designed 3D structure. When evenly mixed with bioink, stem cells can be spatially distributed within a bioprinted 3D structure, thus avoiding drawbacks such as, stem cell detachment in a conventional cell-scaffold strategy. Notwithstanding the advantages mentioned above, cell viability is often compromised during 3D bioprinting, which is often due to pressure during the bioprinting process. The objective of our study was to improve the efficiency of hADSC smooth muscle differentiation and cell viability of a 3D bioprinted structure. Here, we employed the hanging-drop method to generate hADSC microtissues in a smooth muscle inductive medium containing human transforming growth factor β1 and bioprinted the induced microtissues onto a 3D structure. After 3 days of smooth muscle induction, the expression of α-smooth muscle actin and smoothelin was higher in microtissues than in their counterpart monolayer cultured hADSCs, as confirmed by immunofluorescence and western blotting analysis. The semi-quantitative assay showed that the expression of α-smooth muscle actin (α-SMA) was 0.218 ± 0.077 in MTs and 0.082 ± 0.007 in Controls; smoothelin expression was 0.319 ± 0.02 in MTs and 0.178 ± 0.06 in Controls. Induced MTs maintained their phenotype after the bioprinting process. Live/dead and cell count kit 8 assays showed that cell viability and cell proliferation in the 3D structure printed with microtissues were higher at all time points compared to the conventional single-cell bioprinting strategy (mean cell viability was 88.16 ± 3.98 vs. 61.76 ± 15% for microtissues and single-cells, respectively). These results provide a novel way to enhance the smooth muscle differentiation of hADSCs and a simple method to maintain better cell viability in 3D bioprinting.

Functional characterization of 3D contractile smooth muscle tissues generated using a unique microfluidic 3D bioprinting technology

“…35 Implanted skeletal muscle tissue has shown an ability to increase force generation of damaged muscles. 38 Our work adds to the field of 3D muscle cell biology by demonstrating the benefits of 3D bioprinting of smooth muscle cells for studying contractile responses and further illustrates that the use of dECM can extend to functional outputs such as contraction, relaxation, and drug responses in smooth muscle cells. 37 Adipose-derived stem cells cultured in 3D were more able than those cultured in 2D to differentiate into smooth muscle cells, as determined by expression of smooth muscle actin and smoothelin.…”

“…37 Adipose-derived stem cells cultured in 3D were more able than those cultured in 2D to differentiate into smooth muscle cells, as determined by expression of smooth muscle actin and smoothelin. 38 Our work adds to the field of 3D muscle cell biology by demonstrating the benefits of 3D bioprinting of smooth muscle cells for studying contractile responses and further illustrates that the use of dECM can extend to functional outputs such as contraction, relaxation, and drug responses in smooth muscle cells.…”

3D Bioprinting Stem Cell Derived Tissues

“…Another group bioprinted hADSC-derived smooth muscle cells evenly mixed with a gelatin bioink using an extrusion-based 3D bioprinter. 193 Their results showed enhancement in smooth muscle cell differentiation and maintained more robust cell viability and cell proliferation compared to other conventional bioprinting methods. Higher expression of smooth muscle actin and smoothelin was observed after 3 days and western blotting analysis confirmed the differentiation of smooth muscle cells.…”

Progress in scaffold‐free bioprinting for cardiovascular medicine

“…Another attractive feature of scaffold-free bioprinting is its efficiency, as the speed can be comparable or even higher than other forms of bioprinting by using as "building blocks" large spheroids (in the tens of cellular cross-talk proceeds naturally, while optional addition of hydrogels in between the cells and within spheroids still remains possible and probably beneficial. 26,27 Thus, since scalability is more limited, scaffold-free-methods are preferable for smaller, cell-heterogeneous, matrix-poor tissues where the immediate (or continuous) intercellular communication is important.…”