Cellular Stemness Maintenance of Human Adipose‐Derived Stem Cells on ZnO Nanorod Arrays

Ever‐growing tissue regeneration and other stem cell therapies cause pressing need for large population of self‐renewable stem cells. However, stem cells gradually lose their stemness after long‐term in vitro cultivation. In this study, a ZnO nanorod (ZnO NR) array is used to maintain the stemness of human adipose‐derived stem cells (hADSCs). The results prove that after culturing hADSCs on ZnO NRs for 3 weeks, the stemness genes and protein expression level are higher than that on culture plates and ZnO film. ZnO NRs can maintain stemness of hADSCs without inhibiting the cell proliferation and oriented differentiation capabilities. KLF4 (Kruppel‐like factor 4) is a Zn²⁺‐binding gene that plays a vital role in cell proliferation and differentiation. Sustained Zn²⁺ release and the increased expression of KLF4 can be detected, suggesting that ZnO NRs have efficiently released Zn²⁺ for stemness maintenance. Taken together, the nanotopography of ZnO NRs and the Zn²⁺ release synergistically facilitate stemness maintenance. This study has provided a powerful tool for directing cell fate, maintaining stemness, and realizing the expansion of stem cells in vitro, which will open a new route for the manufacture of large populations of stem cells and fulfilling the growing demand for the cell therapy market.     

Screening Therapeutic Agents Specific to Breast Cancer Stem Cells Using a Microfluidic Single‐Cell Clone‐Forming Inhibition Assay

Screens of cancer stem cells (CSCs)‐specific agents present significant challenges to conventional cell assays due to the difficulty in preparing CSCs ready for drug testing. To overcome this limitation, developed is a microfluidic single‐cell assay for screening breast cancer stem cell–specific agents. This assay takes advantage of the single‐cell clone‐forming capability of CSCs, which can be specifically inhibited by CSC‐targeting agents. The single‐cell assay is performed on a microfluidic chip with an array of 3840 cell‐capturing units; the single‐cell arrays are easily formed by flowing a cell suspension into the microchip. Achieved is a single cell‐capture rate of ≈60% thus allowing more than 2000 single cells to be analyzed in a single test. Over long‐term suspension culture, only a minority of cells survive and form tumorspheres. The clone‐formation rate of MCF‐7, MDA‐MB‐231, and T47D cells is 1.67%, 5.78%, and 5.24%, respectively. The clone‐forming inhibition assay is conducted by exposing the single‐cell arrays to a set of anticancer agents. The CSC‐targeting agents show complete inhibition of single‐cell clone formation while the nontargeting ones show incomplete inhibition effects. The resulting microfluidic single‐cell assay with the potential to screen CSC‐specific agents with high efficiency provides new tools for individualized tumor therapy.     

Mussel Adhesion‐Inspired Reverse Transfection Platform Enhances Osteogenic Differentiation and Bone Formation of Human Adipose‐Derived Stem Cells

Using small interfering RNA (siRNA) to regulate gene expression is an emerging strategy for stem cell manipulation to improve stem cell therapy. However, conventional methods of siRNA delivery into stem cells based on solution‐mediated transfection are limited due to low transfection efficiency and insufficient duration of cell‐siRNA contact during lengthy culturing protocols. To overcome these limitations, a bio‐inspired polymer‐mediated reverse transfection system is developed consisting of implantable poly(lactic‐co‐glycolic acid) (PLGA) scaffolds functionalized with siRNA‐lipidoid nanoparticle (sLNP) complexes via polydopamine (pDA) coating. Immobilized sLNP complexes are stably maintained without any loss of siRNA on the pDA‐coated scaffolds for 2 weeks, likely due to the formation of strong covalent bonds between amine groups of sLNP and catechol group of pDA. siRNA reverse transfection with the pDA‐sLNP‐PLGA system does not exhibit cytotoxicity and induces efficient silencing of an osteogenesis inhibitor gene in human adipose‐derived stem cells (hADSCs), resulting in enhanced osteogenic differentiation of hADSCs. Finally, hADSCs osteogenically committed on the pDA‐sLNP‐PLGA scaffolds enhanced bone formation in a mouse model of critical‐sized bone defect. Therefore, the bio‐inspired reverse transfection system can provide an all‐in‐one platform for genetic modification, differentiation, and transplantation of stem cells, simultaneously enabling both stem cell manipulation and tissue engineering.     

Nondestructive Real‐Time Monitoring of Enhanced Stem Cell Differentiation Using a Graphene‐Au Hybrid Nanoelectrode Array

Stem cells have attracted increasing research interest in the field of regenerative medicine because of their unique ability to differentiate into multiple cell lineages. However, controlling stem cell differentiation efficiently and improving the current destructive characterization methods for monitoring stem cell differentiation are the critical issues. To this end, multifunctional graphene–gold (Au) hybrid nanoelectrode arrays (NEAs) to: (i) investigate the effects of combinatorial physicochemical cues on stem cell differentiation, (ii) enhance stem cell differentiation efficiency through biophysical cues, and (iii) characterize stem cell differentiation in a nondestructive real‐time manner are developed. Through the synergistic effects of physiochemical properties of graphene and biophysical cues from nanoarrays, the graphene‐Au hybrid NEAs facilitate highly enhanced cell adhesion and spreading behaviors. In addition, by varying the dimensions of the graphene‐Au hybrid NEAs, improved stem cell differentiation efficiency, resulting from the increased focal adhesion signal, is shown. Furthermore, graphene‐Au hybrid NEAs are utilized to monitor osteogenic differentiation of stem cells electrochemically in a nondestructive real‐time manner. Collectively, it is believed the unique multifunctional graphene‐Au hybrid NEAs can significantly advance stem‐cell‐based biomedical applications.     

TiO2 Nanorod Array Constructed Nanotopography for Regulation of Mesenchymal Stem Cells Fate and the Realization of Location‐Committed Stem Cell Differentiation

As a physical cue for controlling the fate of stem cells, surface nanotopography has attracted much attention to improve the integration between implants and local host tissues and cells. A biocompatible surface TiO₂ nanorod array is proposed to regulate the fate of bone marrow derived mesenchymal stem cells (MSCs). TiO₂ substrates with different surface nanotopographies: a TiO₂ nanorod array and a polished TiO₂ ceramic are built by hydrothermal and sintering processes, respectively. The assessment of morphology, viability, gene expression, and protein characterization of the MSCs cultured on the different TiO₂ substrates proves that a TiO₂ nanorod array promotes the osteogenic differentiation of MSCs, while a TiO₂ ceramic with a smooth surface suppresses it. Periodically assembled TiO₂ nanorod array stripes on the smooth TiO₂ ceramic are constructed by a combination of microfabrication and a chemical synthesis process, which realizes the location‐committed osteogenic differentiation of MSCs. A route to control the differentiation of MSCs by a nanostructured surface, which can also control the location and direction of MSCs on the surface of biomaterials with micro‐nano scale surface engineering, is demonstrated.     

Inertial Microfluidic Cell Stretcher (iMCS): Fully Automated,High‐Throughput,and Near Real‐Time Cell Mechanotyping

Mechanical biomarkers associated with cytoskeletal structures have been reported as powerful label‐free cell state identifiers. In order to measure cell mechanical properties, traditional biophysical (e.g., atomic force microscopy, micropipette aspiration, optical stretchers) and microfluidic approaches were mainly employed; however, they critically suffer from low‐throughput, low‐sensitivity, and/or time‐consuming and labor‐intensive processes, not allowing techniques to be practically used for cell biology research applications. Here, a novel inertial microfluidic cell stretcher (iMCS) capable of characterizing large populations of single‐cell deformability near real‐time is presented. The platform inertially controls cell positions in microchannels and deforms cells upon collision at a T‐junction with large strain. The cell elongation motions are recorded, and thousands of cell deformability information is visualized near real‐time similar to traditional flow cytometry. With a full automation, the entire cell mechanotyping process runs without any human intervention, realizing a user friendly and robust operation. Through iMCS, distinct cell stiffness changes in breast cancer progression and epithelial mesenchymal transition are reported, and the use of the platform for rapid cancer drug discovery is shown as well. The platform returns large populations of single‐cell quantitative mechanical properties (e.g., shear modulus) on‐the‐fly with high statistical significances, enabling actual usages in clinical and biophysical studies.     

Surface‐Acoustic‐Wave‐Based Lab‐on‐Chip for Rapid Transport of Cryoprotectants across Cell Membrane for Cryopreservation with Significantly Improved Cell Viability

Cryopreservation is essential to effectively extend the shelf life of delicate biomaterials while maintaining proper levels of cell functions. Cryopreservation requires a cryoprotective agent (CPA) to suppress intracellular ice formation during freezing, but it must be removed prior to clinical use due to its toxicity. Conventional multistep CPA loading and unloading approaches are time consuming, often creating osmotic shocks and causing mechanical injuries for biological samples. An efficient surface‐acoustic‐wave‐ (SAW‐) based lab‐on‐a‐chip (LoC) for fast loading and removal of CPAs is presented here. With the SAW‐based multistep CPA loading/removal approach, high concentration (3 m ) CPA can be successfully loaded and removed in less than 1 min. Results show that the technique causes the least harm to umbilical cord matrix mesenchymal stem cells as compared to conventional method, and an average of 24% higher cell recovery rate is achieved, while preserving the integrity and morphology of the cells. This device is the first of its kind to combine high loading/unloading efficiency, high cell viability, and high throughput into one LoC device, offering not only a more efficient and safer route for CPA loading and removal from cells, but also paving the way for other cryopreservation‐dependent applications.     

In Situ Real‐Time Study of the Dynamic Formation and Conversion Processes of Metal Halide Perovskite Films

Metal halide perovskite solar cells (PSCs) have advanced to the forefront of solution‐processed photovoltaic techniques and made stunning progress in power conversion efficiency (PCE). Further improvements in device performances rely on perfecting the structure and morphology of perovskite films. However, undesirable defects such as pinholes and grain boundaries are often created in film preparations due to lack of knowledge of the precise reaction mechanism. Here, in situ grazing‐incidence X‐ray diffraction (GI‐XRD) investigations are performed, facilitated by other techniques, on the formation of the widely adopted MAPbI₃ (MA = methylammonium) perovskite films from their intermediate adduct (IA) phases. The influences of solvent vapor atmospheres on MAPbI₃ films are also systematically investigated, where the dynamic conversion processes between different phases are visualized in real time. Further in situ GI‐XRD and infrared spectroscopy measurements reveal that the IA phases contain both N,N‐dimethylformamide and dimethyl sulfoxide (DMSO) as coordinating molecules. By tuning the DMSO concentration in perovskite precursors, the ideal perovskite film is formed and the best PCE is achieved for the planar MAPbI₃‐based PSCs. These findings highlight the role of IA phases and the effect of solvent atmospheres on the quality of perovskite films, providing direct insights into their growth mechanism.     

Mechanical Cues Regulating Proangiogenic Potential of Human Mesenchymal Stem Cells through YAP‐Mediated Mechanosensing

Stem cells secrete trophic factors that induce angiogenesis. These soluble factors are promising candidates for stem cell–based therapies, especially for cardiovascular diseases. Mechanical stimuli and biophysical factors presented in the stem cell microenvironment play important roles in guiding their behaviors. However, the complex interplay and precise role of these cues in directing pro‐angiogenic signaling remain unclear. Here, a platform is designed using gelatin methacryloyl hydrogels with tunable rigidity and a dynamic mechanical compression bioreactor to evaluate the influence of matrix rigidity and mechanical stimuli on the secretion of pro‐angiogenic factors from human mesenchymal stem cells (hMSCs). Cells cultured in matrices mimicking mechanical elasticity of bone tissues in vivo show elevated secretion of vascular endothelial growth factor (VEGF), one of representative signaling proteins promoting angiogenesis, as well as increased vascularization of human umbilical vein endothelial cells (HUVECs) with a supplement of conditioned media from hMSCs cultured across different conditions. When hMSCs are cultured in matrices stimulated with a range of cyclic compressions, increased VEGF secretion is observed with increasing mechanical strains, which is also in line with the enhanced tubulogenesis of HUVECs. Moreover, it is demonstrated that matrix stiffness and cyclic compression modulate secretion of pro‐angiogenic molecules from hMSCs through yes‐associated protein activity.