Highly stable, non-living biological engineered mesenchymal stem cell-based reservoirs enable the manufacture of disease-specific formulations for regenerative medicine applications and to overcome complex fabrication processes.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by impaired barrier function and helper T cell 2 (Th2)-dominant immune dysregulation. Subcutaneous administration of mesenchymal-stem-cell-derived extracellular vesicles (MSC-EVs) has shown substantial efficacy in preclinical AD models; however, its clinical utility remains restricted by injection-associated pain and poor patient compliance, necessitating more patient-friendly delivery strategies. Here, we propose a patient-friendly, noninvasive therapeutic strategy using clinical-grade MSC-EVs incorporated into a thermosensitive hyaluronic acid/Pluronic F127 (HP) hydrogel. The optimized HP hydrogel exhibited desirable shear-thinning behavior and tunable sol-gel transition at body temperature, enabling sustained EV release. In a Dermatophagoides farinae-extract-induced AD mouse model, topical application of HP-loaded EVs (HP+EVs) suppressed AD symptoms, epidermal thickening, and mast cell infiltration, demonstrating an efficacy comparable to that of subcutaneous EV injections. Notably, HP+EV treatment down-regulated both Th2 and Th1/pro-inflammatory cytokines, restoring their levels to those in healthy controls. In addition, EVs rapidly penetrated the disrupted skin barrier in AD lesions via surface-protein-dependent mechanisms, as evidenced by the loss of permeability following proteinase K treatment. Furthermore, HP+EV treatment up-regulated barrier proteins and markedly down-regulated oxidative stress markers. This study demonstrates a major advancement in EV-based therapeutics, providing a promising noninvasive formulation for the treatment of AD.

Stem-cell-derived extracellular vesicles (EVs) have emerged as a promising therapeutic option, addressing the limitations of conventional stem cell therapies. However, the variability and poorly defined therapeutic contents of EVs produced under standard 2-dimensional culture conditions present challenges for their clinical application. In this study, we investigated how the therapeutic properties of mesenchymal stem cell (MSC)-derived EVs can be enhanced by culturing MSCs within 3-dimensional hydrogels that have tunable mechanical properties. Our results demonstrate that different mechanical cues from the culture environment can induce specific gene expression changes in MSCs without compromising their inherent characteristics. Furthermore, EVs derived from these MSCs exhibited distinct angiogenic and immunomodulatory activities, which were dependent on the mechanical properties of the hydrogels used. A comprehensive analysis of the cytokines and microRNAs present in the EVs provided additional validation of these findings. By utilizing a noninvasive culture method that eliminates the need for genetic modification or exogenous biochemical supplementation, our approach presents a novel platform for the tailored production of EVs, thereby enhancing their therapeutic potential in regenerative medicine.

Stem-cell-derived extracellular vesicles (EVs) are emerging as an alternative approach to stem cell therapy. Successful lyophilization of EVs could enable convenient storage and distribution of EV medicinal products at room temperature for long periods, thus considerably increasing the accessibility of EV therapeutics to patients. In this study, we aimed to identify an appropriate lyoprotectant composition for the lyophilization and reconstitution of stem-cell-derived EVs. MSC-derived EVs were lyophilized using different lyoprotectants, such as dimethyl sulfoxide, mannitol, trehalose, and sucrose, at varying concentrations. Our results revealed that a mixture of trehalose and sucrose at high concentrations could support the formation of amorphous ice by enriching the amorphous phase of the solution, which successfully inhibited the acceleration of buffer component crystallization during lyophilization. Lyophilized and reconstituted EVs were thoroughly evaluated for concentration and size, morphology, and protein and RNA content. The therapeutic effects of the reconstituted EVs were examined using a tube formation assay with human umbilical vein endothelial cells. After rehydration of the lyophilized EVs, most of their generic characteristics were well-maintained, and their therapeutic capacity recovered to levels similar to those of freshly collected EVs. The concentrations and morphologies of the lyophilized EVs were similar to the initial features of the fresh EV group until day 30 at room temperature, although their therapeutic capacity appeared to decrease after 7 days. Our study suggests an appropriate composition of lyoprotectants, particularly for EV lyophilization, which could encourage the applications of stem-cell-derived EV therapeutics in the health industry.

A major clinical hurdle to translate MSC-derived extracellular vesicles (EVs) is the lack of a method to scale-up the production of EVs with customized therapeutic properties. In this study, we tested whether EV production by a scalable 3D-bioprocessing method is feasible and improves neuroplasticity in animal models of stroke using MRI study. MSCs were cultured in a 3D-spheroid using a micro-patterned well. The EVs were isolated with filter and tangential flow filtration and characterized using electron microscopy, nanoparticle tracking analysis, and small RNA sequencing. Compared to conventional 2D culture, the production-reproduction of EVs (the number/size of particles and EV purity) obtained from 3D platform were more consistent among different lots from the same donor and among different donors. Several microRNAs with molecular functions associated with neurogenesis were upregulated in EVs obtained from 3D platform. EVs induced both neurogenesis and neuritogenesis via microRNAs (especially, miR-27a-3p and miR-132-3p)-mediated actions. EV therapy improved functional recovery on behavioral tests and reduced infarct volume on MRI in stroke models. The dose of MSC-EVs of 1/30 cell dose had similar therapeutic effects. In addition, the EV group had better anatomical and functional connectivity on diffusion tensor imaging and resting-state functional MRI in a mouse stroke model. This study shows that clinical-scale MSC-EV therapeutics are feasible, cost-effective, and improve functional recovery following experimental stroke, with a likely contribution from enhanced neurogenesis and neuroplasticity.

Background: As stem cells are considered a promising cell source for tissue engineering, many culture strategies have been extensively studied to generate in vitro stem cell-based tissue constructs. However, most approaches using conventional tissue culture plates are limited by the lack of biological relevance in stem cell microenvironments required for neotissue formation. In this study, a novel perfusion rotating wall vessel (RWV) bioreactor was developed for mass-production of stem cell-based 3D tissue constructs. Methods: An automated RWV bioreactor was fabricated, which is capable of controlling continuous medium perfusion, highly efficient gas exchange with surrounding air, as well as low-intensity pulsed ultrasound (LIPUS) stimulation. Embryonic stem cells encapsulated in alginate/gelatin hydrogel were cultured in the osteogenic medium by using our bioreactor system. Cellular viability, growth kinetics, and osteogenesis/mineralization were thoroughly evaluated, and culture media were profiled at real time. The in vivo efficacy was examined by a rabbit cranial defect model. Results: Our bioreactor successfully maintained the optimal culture environments for stem cell proliferation, osteogenic differentiation, and mineralized tissue formation during the culture period. The mineralized tissue constructs produced by our bioreactor demonstrated higher void filling efficacy in the large bone defects compared to the group implanted with hydrogel beads only. In addition, the LIPUS modules mounted on our bioreactor successfully reached higher mineralization of the tissue constructs compared to the groups without LIPUS stimulation. Conclusion: This study suggests an effective biomanufacturing strategy for mass-production of implantable mineralized tissue constructs from stem cells that could be applicable to future clinical practice.

Spinal fusion surgery is a surgical technique that connects one or more vertebrae at the same time to prevent movement between the vertebrae. Although synthetic bone substitutes or osteogenesis-inducing recombinant proteins were introduced to promote bone union, the rate of revision surgery is still high due to pseudarthrosis. To promote successful fusion after surgery, stem cells with or without biomaterials were introduced; however, conventional 2D-culture environments have resulted in a considerable loss of the innate therapeutic properties of stem cells. Therefore, we conducted a preclinical study applying 3D-spheroids of human bone marrow-dewrived mesenchymal stem cells (MSCs) to a mouse spinal fusion model. First, we built a large-scale manufacturing platform for MSC spheroids, which is applicable to good manufacturing practice (GMP). Comprehensive biomolecular examinations, which include liquid chromatography-mass spectrometry and bioinformatics could suggest a framework of quality control (QC) standards for the MSC spheroid product regarding the identity, purity, viability, and potency. In our animal study, the mass-produced and quality-controlled MSC spheroids, either undifferentiated or osteogenically differentiated were well-integrated into decorticated bone of the lumbar spine, and efficiently improved angiogenesis, bone regeneration, and mechanical stability with statistical significance compared to 2D-cultured MSCs. This study proposes a GMP-applicable bioprocessing platform and QC directions of MSC spheroids aiming for their clinical application in spinal fusion surgery as a new bone graft substitute.

Digital-light-processing (DLP) three-dimensional (3D) bioprinting, which has a rapid printing speed and high precision, requires optimized biomaterial ink to ensure photocrosslinking for successful printing. However, optimization studies on DLP bioprinting have yet to sufficiently explore the measurement of light exposure energy and biomaterial ink absorbance controls to improve the printability. In this study, we synchronized the light wavelength of the projection base printer with the absorption wavelength of the biomaterial ink. In this paper, we provide a stepwise explanation of the challenges associated with unsynchronized absorption wavelengths and provide appropriate examples. In addition to biomaterial ink wavelength synchronization, we introduce photorheological measurements, which can provide optimized light exposure conditions. The photorheological measurements provide precise numerical data on light exposure time and, therefore, are an effective alternative to the expendable and inaccurate conventional measurement methods for light exposure energy. Using both photorheological measurements and bioink wavelength synchronization, we identified essential printability optimization conditions for DLP bioprinting that can be applied to various fields of biological sciences.

181Biofabrication approaches, such as three-dimensional (3D) bioprinting of hydrogels, have recently garnered increasing attention, especially in the construction of 3D structures that mimic the complexity of tissues and organs with the capacity for cytocompatibility and post-printing cellular development. However, some printed gels show poor stability and maintain less shape fidelity if parameters such as polymer nature, viscosity, shear-thinning behavior, and crosslinking are affected. Therefore, researchers have incorporated various nanomaterials as bioactive fillers into polymeric hydrogels to address these limitations. Carbon-family nanomaterials (CFNs), hydroxyapatites, nanosilicates, and strontium carbonates have been incorporated into printed gels for application in various biomedical fields. In this review, following the compilation of research publications on CFNs-containing printable gels in various tissue engineering applications, we discuss the types of bioprinters, the prerequisites of bioink and biomaterial ink, as well as the progress and challenges of CFNs-containing printable gels in this field.

The development of biosensors that measure various biosignals from our body is an indispensable research field for health monitoring. In recent years, as the demand to monitor the health conditions of individuals in real time have increased, wearable-type biosensors have received more attention as an alternative to laboratory equipment. These biosensors have been embedded into smart watches, clothes, and accessories to collect various biosignals in real time. Although wearable biosensors attached to the human body can conveniently collect biosignals, there are reliability issues due to noise generated in data collection. In order for wearable biosensors to be more widely used, the reliability of collected data should be improved. Research on flexible bio-chips in the field of material science and engineering might help develop new types of biosensors that resolve the issues of conventional wearable biosensors. Flexible bio-chips with higher precision can be used to collect various human data in academic research and in our daily lives. In this review, we present various types of conventional biosensors that have been used and discuss associated issues such as noise and inaccuracy. We then introduce recent studies on flexible bio-chips as a solution to these issues.

Bioink is the main component of 3D bioprinting process and is crucial for the generation of sophisticated 3D structures through precise spatial control. Therefore, bioink’s core material must have characteristics that support good printability as well as biocompatibility. However, there is a lack of bioinks developed that satisfy these characteristics at the same time. In this work, our aim was to develop a bioink that satisfies the needs for both printability and biocompatibility through effectively utilizing hydrocolloid materials. To do so, carboxymethyl cellulose (CMC) and xanthan gum (XG) were used to maintain proper shear properties at high pressure and increase the mechanical properties of bioink without excessively affecting the viscosity, and thus enhance printability and biocompatibility. Various bioink formulations were applied to 3D printing process and the printability optimization was carried out through adjusting the hydrocolloid contents in connection with different cross-linking methods. Through utilization of hydrocolloids, the printability and rheological analysis showed that the bioink has improved mechanical properties and confirmed that the printability could be adjusted by controlling the CMC and XG ratio. Moreover, cell viability and immunocytochemical staining analyses showed cell compatibility with enhanced stability. The proposed convenient method to control the printability with improved biocompatibility suggests more appropriate use of bioink for co-axial 3D bioprinting.

Over the past decades, a number of bone tissue engineering (BTE) approaches have been developed to address substantial challenges in the management of critical size bone defects. Although the majority of BTE strategies developed in the laboratory have been limited due to lack of clinical relevance in translation, primary prerequisites for the construction of vascularized functional bone grafts have gained confidence owing to the accumulated knowledge of the osteogenic, osteoinductive, and osteoconductive properties of mesenchymal stem cells and bone-relevant biomaterials that reflect bone-healing mechanisms. In this review, we summarize the current knowledge of bone-healing mechanisms focusing on the details that should be embodied in the development of vascularized BTE, and discuss promising strategies based on 3D-bioprinting technologies that efficiently coalesce the abovementioned main features in bone-healing systems, which comprehensively interact during the bone regeneration processes.

Thermo-sensitive injectable hydrogels that spontaneously react to physiological temperature have been widely studied to be used in biomedical fields. However, several challenges on their unstable structures with large-sized pores and low mechanical strength under physiological conditions must be addressed to enable their practical applications. We synthesized the hydroxybutyl methacrylated chitosan (HBC-MA) hydrogel that possesses both thermo-sensitive and photo-crosslinkable properties. The HBC-MA showed effective sol-gel transition under physiological temperature as well as a sensitive photo-crosslinkable property with visible light capable of skin penetration. The co-nonsolvency property and thermo-sensitivity of HBC-MA prevented unintended loss of the hydrogel graft after being subcutaneously injected in mice. Subsequently applied visible light on the skin beneath which the hydrogel was injected significantly improved the mechanical strength and stability of the graft. The injectable HBC-MA hydrogel developed in this study can be applicable to a wide range of biomedical fields such as drug delivery system and tissue engineering.

Bioink based 3D bioprinting is a promising new technology that enables fabrication of complex tissue structures with living cells. The printability of the bioink depends on the physical properties such as viscosity. However, the high viscosity bioink puts shear stress on the cells and low viscosity bioink cannot maintain complex tissue structure firmly after the printing. In this work, we applied dual crosslinkable bioink using Kappa-carrageenan (κ-CA) to overcome existing shortcomings. κ-CA has properties such as biocompatibility, biodegradability, shear-thinning and ionic gelation but the difficulty of controlling gelation properties makes it unsuitable for application in 3D bioprinting. This problem was solved by synthesizing methacrylated Kappa-carrageenan (MA-κ-CA), which can be dual crosslinked through ionic and UV (Ultraviolet) crosslinking to form hydrogel using NIH-3T3 cells. Through MA substitutions, the rheological properties of the gel could be controlled to reduce the shear stress. Moreover, bioprinting using the cell-laden MA-κ-CA showed cell compatibility with enhanced shape retention capability. The potential to control the physical properties through dual crosslinking of MA-κ-CA hydrogel is expected to be widely applied in 3D bioprinting applications.

The gelatin extracted from mammals of porcine and bovine has been prominently used in pharmaceutical, medical, and cosmetic products. However, there have been some concerns for their usage due to religious, social and cultural objections, and animal-to-human infectious disease. Recently, gelatin from marine by-products has received growing attention as an alternative to mammalian gelatin. In this study, we demonstrate the formation of nanogels (NGs) using fish gelatin methacryloyl (GelMA) and their application possibility to the drug delivery system. The fabrication of fish GelMA NGs is carried out by crosslinking through the photopolymerization of the methacryloyl substituent present in the nanoemulsion droplets, followed by purification and redispersion. There were different characteristics depending on the aqueous phase in the emulsion and the type of solvent used in redispersion. The PBS-NGs/D.W., which was prepared using PBS for the aqueous phase and D.W. for the final dispersion solution, had a desirable particle size (<200 nm), low PdI (0.16), and high drug loading efficiency (77%). Spherical NGs particles were observed without aggregation in TEM images. In vitro release tests of doxorubicin (DOX)-GelMA NGs showed the pH-dependent release behavior of DOX. Also, the MTT experiments demonstrated that DOX-GelMA NGs effectively inhibited cell growth, while only GelMA NGs exhibit higher percentages of cell viability. Therefore, the results suggest that fish GelMA NGs have a potential for nano-carrier as fine individual particles without the aggregation and cytotoxicity to deliver small-molecule drugs.

Mesenchymal stem cells (MSCs) exert their therapeutic capability through a variety of bioactive substances, including trophic factors, microRNAs, and extracellular vesicles (EVs) in infarcted tissues. We therefore hypothesized that MSC-derived EVs (MSC-EVs) possess therapeutic molecules similar to MSCs. Moreover, given their nature as nanosized and lipid-shielded particles, the intravenous infusion of MSC-EVs would be advantageous over MSCs as a safer therapeutic approach. In this study, we investigated the biodistribution, therapeutic efficacy, and mode of action of MSC-EVs in a rat stroke model. MSC-EVs successfully stimulated neurogenesis and angiogenesis in vivo. When compared to the MSC-treated group, rats treated with MSC-EVs exhibited greater behavioral improvements than the control group (p < 0.05). Our biodistribution study using fluorescence-labeled MSC-EVs and MSCs demonstrated that the amounts of MSC-EVs in the infarcted hemisphere increased in a dose-dependent manner, and were rarely found in the lung and liver. In addition, MSC-EVs were highly inclusive of various proteins and microRNAs (miRNAs) associated with neurogenesis and/or angiogenesis compared to fibro-EVs. We further analyzed those miRNAs and found that miRNA-184 and miRNA-210 were essential for promoting neurogenesis and angiogenesis of MSC-EVs, respectively. MSC-EVs represent an ideal alternative to MSCs for stroke treatment, with similar medicinal capacity but an improved safety profile that overcomes cell-associated limitations in stem cell therapy.

The procedures and protocols for the pre-treatment of sputum specimens, mainly used for the diagnosis of pneumonia, are complex, labor intensive, and require skilled specialists working in a biosafety containment laboratory because of sample infectivity. In this study, we developed the first portable, low-power pre-treatment device that carries out all sputum pre-treatment procedures (liquefaction, homogenization, dissolution, and inactivation) in an enclosed space. Designed to simultaneously employ chemical and mechanical dissolution in the enclosed chamber, this device eliminates the risk of transmission and improves the effectiveness of sputum dissolution and pathogen detection. This device is expected to allow for the pre-treatment of infectious sputum specimens outside of a biosafety containment laboratory. Used in conjunction with automated genome extraction and detection systems, this device should make the on-site diagnosis using infectious sputum specimens possible.

As stem cell technologies have rapidly advanced, the stem cell therapy market has been forecast to reach hundreds of millions of USD in market value within the next 5 years. Regulatory frameworks throughout the stem cell market have been concurrently established, which will encourage the advent of a variety of stem cell products in our society. Given the circumstances, stem cell bioprocessing has emerged as one of the most critical fields of research to address a number of issues that currently exist in manufacturing clinical-grade stem cells at an industrial scale. Highly specialized bioreactor designs are at the center of essentially required technologies in the field of stem cell bioprocessing, which ultimately aim for automated, standardized, traceable, cost-effective, safe, and regulatory-compliant manufacture of stem cell-based products. In this review, recently developed bioreactor designs to introduce important regulatory factors to three-dimensional stem cell culture are exemplified, and prerequisites for the ideal bioreactor systems for stem cell bioprocessing are discussed.