Enhancing the production of adeno-associated virus (AAV)2 and AAV9 with high full capsid ratio in HEK293 cells through design-of-experiment optimization of triple plasmid ratio.

The recombinant adeno-associated virus (rAAV) vectors used in gene therapy are usually produced by transfecting three different plasmids (Adenoviral helper plasmid (pHelper), AAV rep/cap plasmids (pRepCap), and Transgene plasmid (pAAV-GOI)) into human embryonic kidney 293 (HEK293) cells. However, the high proportion of unwanted empty capsids generated during rAAV production is problematic. To simultaneously enhance the genome titer and full capsid ratio, the ratio of the three plasmids transfected into HEK293 cells was optimized using design-of-experiment (DoE). AAV2 and AAV9, which have different production kinetics, were selected as cell-associated and secreted model AAVs, respectively. In 125 mL Erlenmeyer flasks, the genome titers of rAAV2 and rAAV9 at DoE-optimized plasmid weight ratios (pHelper:pRep2Cap2:pAAV-GOI = 1:3.52:0.50 for rAAV2 and pHelper:pRep2Cap9:pAAV-GOI = 1:1.44:0.27 for rAAV9) were 2.23-fold and 2.26-fold higher than those in the widely used plasmid weight ratio (1:1:1), respectively. In addition, compared with the plasmid ratio of 1:1:1, the relative VP3 band intensities of rAAV2 and rAAV9, which represent the relative empty capsid ratios, were reduced by 26% and 25%, respectively, at the DoE-optimized plasmid ratio. Reduced empty capsid ratios in the DoE-optimized plasmid ratios were also confirmed using transmission electron microscopy (TEM). Taken together, regardless of the AAV serotype, DoE-aided optimization of the triple plasmid ratio was found to be an efficient means of improving the production of rAAV with a high full capsid ratio.

Simultaneous coupling of metal removal and visual detection by nature-inspired polyphenol-amine surface chemistry.

The interplay between polyphenols, amines, and metals has broad implications for surface chemistry, biomaterials, energy storage, and environmental science. Traditionally, polyphenol-amine combinations have been recognized for their ability to form adhesive, material-independent thin layers that offer a diverse range of surface functionalities. Herein, we demonstrate that a coating of tannic acid (TA) and polyethyleneimine (PEI) provides an efficient platform for capturing and monitoring metal ions in water. A unique feature of our PEI/TA-coated microbeads is the 'Detection-Capture' (Detec-Ture) mechanism. The galloyl groups in TA coordinate with Fe(III) ions (capture), initiating their oxidation to gallol-quinone. These oxidized groups subsequently react with PEI amines, leading to the formation of an Fe(II/III)-gallol-PEI network that produces a vivid purple color, thereby enabling visual detection. This mechanism couples metal capture directly with detection, distinguishing our approach from existing studies, which have either solely focused on metal removal or metal detection. The metal capturing capacity of our materials stands at 0.55 mg g-1, comparable to that of established materials like alginate and wollastonite. The detection sensitivity reaches down to 0.5 ppm. Our findings introduce a novel approach to the utility of metal-polyphenol-amine networks, presenting a new class of materials suited for simultaneous metal ion detection and capture in environmental applications.

Coagulant Protein-Free Blood Coagulation Using Catechol-Conjugated Adhesive Chitosan/Gelatin Double Layer.

Since the discovery of polyphenolic underwater adhesion in marine mussels, researchers strive to emulate this natural phenomenon in the development of adhesive hemostatic materials. In this study, bio-inspired hemostatic materials that lead to pseudo-active blood coagulation, utilizing traditionally passive polymer matrices of chitosan and gelatin are developed. The two-layer configuration, consisting of a thin, blood-clotting catechol-conjugated chitosan (CHI-C) layer and a thick, barrier-functioning gelatin (Geln) ad-layer, maximizes hemostatic capability and usability. The unique combination of coagulant protein-free condition with CHI-C showcases not only coagulopathy-independent blood clotting properties (efficacy) but also exceptional clinical potential, meeting all necessary biocompatibility evaluation (safety) without inclusion of conventional coagulation triggering proteins such as thrombin or fibrinogen. As a result, the CHI-C/Geln is approved by the Ministry of Food and Drug Safety (MFDS, Republic of Korea) as a class II medical device. Hemostatic efficacy observed in multiple animal models further demonstrates the superiority of CHI-C/Geln sponges in achieving quick hemostasis compared to standard treatments. This study not only enriches the growing body of research on mussel-inspired materials but also emphasizes the potential of biomimicry in developing advanced medical materials, contributing a promising avenue toward development of readily accessible and affordable hemostatic materials.

Low-Viscous, Dilute Phase Adhesive from Dense Polyphenolic Coacervates of Poly(vinyl alcohol) and Tannic acid.

This study explores a polyphenolic coacervate, named VATA, formed by poly(vinyl alcohol) (PVA) and tannic acid (TA). Distinct from conventional studies that have focused on the bottom, dense phase of coacervates, this research emphasizes the top, dilute phase, low-viscous coacervate liquid termed liquid-VATA (l-VATA). Due to TA's capability of intermolecular association as well as adhesiveness, phenomena not typically observed in the upper dilute phase of standard polyelectrolyte-based coacervates are revealed. At first glance, the dilute phase l-VATA coacervate resembles a water-like, low-viscous mixture solution of PVA, TA, and PVA/TA complexes. However, analysis shows that nearly all of the TA molecules associate with PVA chains, forming PVA/TA complexes. Furthermore, supraparticular association was observed between PVA/TA complex nanoparticles upon applying external shear force. A broad survey of shear rate and strain showed that the solution exhibited sequential shear-thickening, followed by shear-thinning behavior. The water-like, low viscosity of l-VATA unexpectedly reveals robust adhesiveness and thus able to lift an entire mouse using just a single human hair strand. Even in cases of failure, no interfacial failure was detected between mouse and human hair. In addition to enabling hair-to-hair bonding, our study also showcases the efficacy of l-VATA in facilitating hair-to-skin adhesion. The results illustrate how the lower viscosity of l-VATA can be exploited for a wide range of industrial and cosmetic applications, allowing the formulation of thin, uniform adhesive layers, something unachievable with the dense, viscous VATA glue. Thus, this study highlights the importance of investigating the top dilute phase of coacervates, shedding light on an area often underestimated compared to the bottom dense phase reported in prevalent coacervate studies.

In vivo genome editing using 244-cis LNPs and low-dose AAV achieves therapeutic threshold in hemophilia A mice.

Gene therapy and rebalancing therapy have emerged as promising approaches for treating hemophilia A, but there are limitations, such as temporary efficacy due to individual differences. Genome editing for hemophilia has shown long-term therapeutic potential in preclinical trials. However, a cautious approach is necessary because genome editing is irreversible. Therefore, we attempted to induce low-level human factor 8 (hF8) gene knockin (KI) using 244-cis lipid nanoparticles and low-dose adeno-associated virus to minimize side effects and achieve a therapeutic threshold in hemophilia A mice. We selected the serpin family C member 1, SerpinC1, locus as a target to enable a combined rebalancing strategy with hF8 KI to augment efficacy. This strategy improved blood coagulation activity and reduced hemophilic complications without adverse effects. Furthermore, hemophilic mice with genome editing exhibit enhanced survival for 40 weeks. Here, we demonstrate an effective, safe, and sustainable treatment for hemophilia A. This study provides valuable information to establish safe and long-term genome-editing-mediated treatment strategies for treating hemophilia and other protein-deficient genetic diseases.

Sniffer worm, C. elegans, as a toxicity evaluation model organism with sensing and locomotion abilities.

Additive manufacturing, or 3D printing, has revolutionized the way we create objects. However, its layer-by-layer process may lead to an increased incidence of local defects compared to traditional casting-based methods. Factors such as light intensity, depth of light penetration, component inhomogeneity, and fluctuations in nozzle temperature all contribute to defect formations. These defective regions can become sources of toxic component leakage, but pinpointing their locations in 3D printed materials remains a challenge. Traditional toxicological assessments rely on the extraction and subsequent exposure of living organisms to these harmful agents, thus only offering a passive detection approach. Therefore, the development of an active system to both identify and locate sources of toxicity is essential in the realm of 3D printing technologies. Herein, we introduce the use of the nematode model organism, Caenorhabditis elegans (C. elegans), for toxicity evaluation. C. elegans exhibits distinctive 'sensing' and 'locomotion' capabilities that enable it to actively navigate toward safe zones while steering clear of hazardous areas. This active behavior sets C. elegans apart from other aquatic and animal models, making it an exceptional choice for immediate and precise identification and localization of toxicity sources in 3D printed materials.

Bioorthogonal CRISPR/Cas9-Drug Conjugate: A Combinatorial Nanomedicine Platform.

Bioconjugation of proteins can substantially expand the opportunities in biopharmaceutical development, however, applications are limited for the gene editing machinery despite its tremendous therapeutic potential. Here, a self-delivered nanomedicine platform based on bioorthogonal CRISPR/Cas9 conjugates, which can be armed with a chemotherapeutic drug for combinatorial therapy is introduced. It is demonstrated that multi-functionalized Cas9 with a drug and polymer can form self-condensed nanocomplexes, and induce significant gene editing upon delivery while avoiding the use of a conventional carrier formulation. It is shown that the nanomedicine platform can be applied for combinatorial therapy by incorporating the anti-cancer drug olaparib and targeting the RAD52 gene, leading to significant anti-tumor effects in BRCA-mutant cancer. The current development provides a versatile nanomedicine platform for combination treatment of human diseases such as cancer.

Ferritin Nanoshuttle for Long-Lasting Self-Healing of Phenolic Hydrogels.

Herein, we highlight a novel finding that ferritin can play a crucial role in the "self-healing lifetime" of soft phenolic materials. Ferritin interacts with a catechol-functionalized polymer to form a self-healable and adhesive hydrogel bidirectionally by providing and retrieving Fe3+. As a result of its unique role as a nanoshuttle to store and release iron, ferritin significantly increases the self-healing lifetime of the hydrogel compared with that afforded by catechol-Fe3+ coordination through direct Fe3+ addition without ferritin. Ferritin also induces stable oxidative coupling between catechol moieties following metal coordination, which contributes to double cross-linking networks of catechol-catechol adducts and catechol-Fe3+ coordination. Thus, ferritin-mediated cross-linking can provide phenolic hydrogels with the advantages of hydrogels prepared by both metal coordination and oxidative coupling, thereby overcoming the limitations of the current cross-linking methods of phenolic hydrogels and broadening their versatility in biomedical applications.

Distinguishing between DNA-Loaded Full and Empty Capsids of Adeno-Associated Virus with Atomic Force Microscopy Imaging.

Recently, miraculous therapy approaches involving adeno-associated virus (AAV) for incurable diseases such as spinal muscular atrophy and inherited retinal dysfunction have been introduced. Nonreplicative, nonpathogenic, low rates of chromosome insertional properties and the existence of neutralizing antibodies are main safety reasons why the FDA approved its use in gene delivery. To date, AAV production always results in a mixture of nontherapeutic (empty) and therapeutic (DNA-loaded) full capsids (10-98%). Such existence of empty viral particles inevitably increases viral doses to human. Thus, the rapid monitoring of empty capsids and reducing the empty-to-full ratio are critical in AAV science. However, transmission electron microscopy (TEM) is the primary tool for distinguishing between empty and full capsids, which creates a research bottleneck because of instrument accessibility and technical difficulty. Herein, we demonstrate that atomic force microscopy (AFM) can be an alternative tool to TEM. The simple, noncontact-mode imaging of AAV particles allows the distinct height difference between full capsids (∼22 nm) and empty capsids (∼16 nm). The sphere-to-ellipsoidal morphological distortion observed for empty AAV particles clearly distinguishes them from full AAV particles. Our study indicates that AFM imaging can be an extremely useful, quality-control tool in AAV particle monitoring, which is beneficial for the future development of AAV-based gene therapy.

Less-Suture Vascular Anastomosis: Development of Alternative Protocols with Multifunctional Self-Wrapping, Transparent, Adhesive, and Elastic Biomaterials.

Blood vessel anastomosis by suture is a life-saving, yet time-consuming and labor-intensive operation. While suture-less alternatives utilizing clips or related devices are developed to address these shortcomings, suture anastomosis is still overwhelmingly used in most cases. In this study, practical "less-suture" strategies are proposed, rather than ideal "suture-less" methods, to reflect real-world clinical situations. In the case of rat artery (d = 0.64 mm) anastomosis, the less-suture anastomosis involves the application of thin, adhesive, transparent, and self-wrapping films to the site. This surprisingly reduces the number of stitches required from ten (without films) to four (with films), saving 27 min of operating time per vessel. Furthermore, the decreased number of stitches largely alleviates fibrosis-mediated wall-thickening. Thus, a less-suture strategy is particularly useful for anastomosis of multiple vessels in emergency conditions and small-diameter vessels.

Phototoxicity-free blue light for enhancing therapeutic angiogenic efficacy of stem cells.

Low-level light therapy (LLLT) is a safe and noninvasive technique that has drawn attention as a new therapeutic method to treat various diseases. However, little is known so far about the effect of blue light for LLLT due to the generation of reactive oxygen species (ROS) that can cause cell damage. We introduced a blue organic light-emitting diode (bOLED) as a safe and effective light source that could generate a low amount of heat and luminance compared to conventional light sources (e.g., light-emitting diodes). We compared phototoxicity of bOLED light with different light fluences to human adipose-derived stem cells (hADSC). We further explored molecular mechanisms involved in the therapeutic efficacy of bOLED for enhancing angiogenic properties of hADSC, including intracellular ROS control in hADSCs. Using optimum conditions of bOLED light proposed in this study, photobiomodulation and angiogenic properties of hADSCs were enhanced. These findings might open new methods for using blue light in LLLT. Such methods can be implemented in future treatments for ischemic disease.

Reversible tissue sticker inspired by chemistry in plant-pathogen relationship.

Plants release phenolic molecules to protect against invading pathogens. In plant-microorganism relationships, phenolics bind to surface oligosaccharides, inactivating microorganism activities. Inspired by phenol-saccharide interactions in plant defense systems, we designed an adhesive sealant. By screening 16 different saccharides, the O-acetyl group, rich in glucomannan (GM), exhibited rapid, robust binding with the galloyl moiety of a model phenolic molecule, tannic acid (TA). Furthermore, the interaction showed both pH and temperature (upper critical solution temperature) sensitivities. Utilizing O-acetyl-galloyl interactions, materials of all dimensions from beads (0D) to strings (1D), films (2D), and objects (3D) could be prepared, as a suitable platform for printing techniques. GMTA films are elastic, adhesive, water-resistant, and effectively sealed perforations, as demonstrated by (1) a lung incision followed by an air inflation model and (2) a thoracic diaphragm model. STATEMENT OF SIGNIFICANCE: In nature, phenolic molecules are 'nearly always' physically bound with polysaccharides, indicating that the phenolics widen the functions of polysaccharides. An example includes that phenolic-polysaccharide interactions are key defense mechanisms against microbial infection in plants whereas polysaccharide alone functions poorly. Despite the ubiquitous biochemistry of polysaccharide-phenolic interactions, efforts on understanding binding chemistry focusing on phenol/polysaccharide interactions is little. This study is important because we found for the first time that O-acetyl group is the moiety in polysaccharides to which phenolic cis-diol and/or cis-triol is spontaneously bound. The phenol-polysaccharide interaction is non-covalent yet robust, kinetically fast, and reversible. Inspired by the interaction chemistry, a simple mixture of phenolic molecules and O-acetyl group containing polysaccharides such as glucomannan opens a promising fabrication strategy toward functional polysaccharide-based material.

Silk Fibroin/Tannin/ZnO Nanocomposite Hydrogel with Hemostatic Activities.

The inevitable bleeding and infections caused by disasters and accidents are the main causes of death owing to extrinsic trauma. Hemostatic agents are often used to quickly suppress bleeding and infection, and they can solve this problem in a short time. Silk fibroin (SF) has poor processibility in water, owing to incomplete solubility therein. In this study, aiming to overcome this disadvantage, a modified silk fibroin (SF-BGE), easily soluble in water, was prepared by introducing butyl glycidyl ether (BGE) into its side chain. Subsequently, a small amount of tannic acid (TA) was introduced to prepare an SF-BGE /TA solution, and ZnO nanoparticles (NPs) were added to the solution to form the coordination bonds between the ZnO and TA, leading to an SF-based nanocomposite hydrogel. A structural characterization of the SF-BGE, SF-BGE/TA, SF-BGE/TA/ZnO, and the coordination bonds between ZnO/TA was observed by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and the phase change was observed by rheological measurements. The pore formation of the SF-BGE/TA/ZnO hydrogel and dispersibility of ZnO were verified through energy-dispersive X-ray spectroscopy (EDS) and scanning electron microscopy (SEM). The cytocompatible and hemostatic performances of the SF-BGE/TA/ZnO NPs composite hydrogels were evaluated, and the hydrogels showed superior hemostatic and cytocompatible activities. Therefore, the SF-based nanocomposite hydrogel is considered as a promising material for hemostasis.

Biodegradable Block Copolymer-Tannic Acid Glue.

Bioadhesives are becoming an essential and important ingredient in medical science. Despite numerous reports, developing adhesive materials that combine strong adhesion, biocompatibility, and biodegradation remains a challenging task. Here, we present a biocompatible yet biodegradable block copolymer-based waterborne superglue that leads to an application of follicle-free hair transplantation. Our design strategy bridges self-assembled, temperature-sensitive block copolymer nanostructures with tannic acid as a sticky and biodegradable polyphenolic compound. The formulation further uniquely offers step-by-step increases in adhesion strength via heating-cooling cycles. Combining the modular design with the thermal treating process enhances the mechanical properties up to 5 orders of magnitude compared to the homopolymer formulation. This study opens a new direction in bioadhesive formulation strategies utilizing block copolymer nanotechnology for systematic and synergistic control of the material's properties.

Mussel-inspired biomaterials: From chemistry to clinic.

After several billions of years, nature still makes decisions on its own to identify, develop, and direct the most effective material for phenomena/challenges faced. Likewise, and inspired by the nature, we learned how to take steps in developing new technologies and materials innovations. Wet and strong adhesion by Mytilidae mussels (among which Mytilus edulis-blue mussel and Mytilus californianus-California mussel are the most well-known species) has been an inspiration in developing advanced adhesives for the moist condition. The wet adhesion phenomenon is significant in designing tissue adhesives and surgical sealants. However, a deep understanding of engaged chemical moieties, microenvironmental conditions of secreted proteins, and other contributing mechanisms for outstanding wet adhesion mussels are essential for the optimal design of wet glues. In this review, all aspects of wet adhesion of Mytilidae mussels, as well as different strategies needed for designing and fabricating wet adhesives are discussed from a chemistry point of view. Developed muscle-inspired chemistry is a versatile technique when designing not only wet adhesive, but also, in several more applications, especially in the bioengineering area. The applications of muscle-inspired biomaterials in various medical applications are summarized for future developments in the field.

Generalized Approach towards Secretion-Based Protein Production via Neutralization of Secretion-Preventing Cationic Substrate Residues.

Many heterologous proteins can be secreted by bacterial ATP-binding cassette (ABC) transporters, provided that they are fused with the C-terminal signal sequence, but some proteins are not secretable even though they carry the right signal sequence. The invention of a method to secrete these non-secretable proteins would be valuable both for understanding the secretory physiology of ABC transporters and for industrial applications. Herein, we postulate that cationic "supercharged" regions within the target substrate protein block the secretion by ABC transporters. We also suggest that the secretion of such substrate proteins can be rescued by neutralizing those cationic supercharged regions via structure-preserving point mutageneses. Surface-protruding, non-structural cationic amino acids within the cationic supercharged regions were replaced by anionic or neutral hydrophilic amino acids, reducing the cationic charge density. The examples of rescued secretions we provide include the spike protein of SARS-CoV-2, glutathione-S-transferase, streptavidin, lipase, tyrosinase, cutinase, growth factors, etc. In summary, our study provides a method to predict the secretability and a tool to rescue the secretion by correcting the secretion-blocking regions, making a significant step in understanding the physiological properties of ABC transporter-dependent protein secretion and laying the foundation for the development of a secretion-based protein-producing platform.

ZnO nanoparticle-embedded modified silk fibroin-tannin multifunctional hydrogel.

Owing to the destruction of ozone layer, the increased exposure to UV on the earth adversely affects not only skin diseases but also wound healing. Although the demand for sunscreens is increasing to protect the human skin from these adverse effects, commercially available sunscreens have some limitations in safety. In this study, silk fibroin (SF) composite with biocompatibility and blood coagulation activity was prepared for a highly safe sunscreen. However, the SF has a disadvantage in that it is difficult to dissolve in water. To improve the solubility of SF, butyl glycidyl ether (BGE) was reacted with the side chain of SF to prepare a freely water-soluble SF (mSF) derivative, and the phase behavior according to the mixing ratio of SF derivative and tannic acid (TA) was observed. In addition, ZnO nanoparticles were added to the mSF-TA solution to form a hydrogel through the coordination bonding. The UV blocking, hemostatic, antibacterial and antioxidant effects of the mSF/TA/ZnO composite hydrogel were evaluated, and the excellent skin compatibility of multifunctional hydrogel sunscreen was confirmed through a skin irritation test.

Addressing the Shortcomings of Polyphenol-Derived Adhesives: Achievement of Long Shelf Life for Effective Hemostasis.

For rapid and effective hemostasis of uncontrollable bleeding, versatile hemostatic agents have been emerging. Among them, polyphenol-derived adhesives have attracted those hemostatic materials due to instantaneous formation of sticky barriers by robust interactions between the material and the serum proteins from wound. However, a critical challenge in such phenolic materials lies in long-term storage due to spontaneous oxidation under humid environments, leading to changes in hemostatic capability and adhesive strength. Here, we report a transparent hemostatic film consisting of gallol-conjugated chitosan (CHI-G) for minimizing the phenolic oxidation even for 3 months and maintaining strong tissue adhesiveness and its hemostatic ability. The film undergoes a phase transition from solid to injectable hydrogels at physiological pH for efficiently stopping internal and external hemorrhage. Interestingly, the hemostatic capability of the CHI-G hydrogels after 3 month storage depends on (i) the folded microstructure of the polymer with optimal gallol modification and (ii) an initial phase of either a solution state or a solid film. When the hydrogels are originated from the dehydrated film, their successful hemostasis is observed in a liver bleeding model. Our finding would provide an insight for design rationale of hemostatic formulations with long shelf-life.

Antagonistically Functionalized Diatom Biosilica for Bio-Triboelectric Generators.

Although biomaterial-based triboelectric nanogenerators (Bio-TENGs) for use in wearable electronics and implantable sensors have been developed, power generation is not suitable for satisfying the basic requirements for practical applications. Here, to greatly enhance output performances of Bio-TENG devices, an antagonistic approach of diatom frustules (DFs) with amine and fluorine chemical functionalizations is reported. The DFs are treated with piranha solution to increase the density of hydroxyl groups and tribo-positive and tribo-negative composite films are designed with antagonistically functionalized DFs. The tribo-positive composites having electron donating functionality consist of aminated DFs and cellulose nanocrystals (CNCs), while the tribo-negative composite is composed of fluorinated DFs and polydimethylsiloxane (PDMS). An antagonistically and chemically functionalized TENG (ACF TENG) with an efficient contact area of 9.6 cm2 under a force of 8 N and a frequency of 5 Hz exhibits an output voltage of 248 V, a short-circuit current of 16.4 µA, and a power density of 2.01 W m-2 , which is 16.6 times higher than a reference (CNC:PDMS) TENG. This study shows a simple antagonistic approach for chemical functionalization as an efficient method to manipulate the tribo-polarity of bio-additives for enhancing power generation of Bio-TENGs.

Area light source-triggered latent angiogenic molecular mechanisms intensify therapeutic efficacy of adult stem cells.

Light-based therapy such as photobiomodulation (PBM) reportedly produces beneficial physiological effects in cells and tissues. However, most reports have focused on the immediate and instant effects of light. Considering the physiological effects of natural light exposure in living organisms, the latent reaction period after irradiation should be deliberated. In contrast to previous reports, we examined the latent reaction period after light exposure with optimized irradiating parameters and validated novel therapeutic molecular mechanisms for the first time. we demonstrated an organic light-emitting diode (OLED)-based PBM (OPBM) strategy that enhances the angiogenic efficacy of human adipose-derived stem cells (hADSCs) via direct irradiation with red OLEDs of optimized wavelength, voltage, current, luminance, and duration, and investigated the underlying molecular mechanisms. Our results revealed that the angiogenic paracrine effect, viability, and adhesion of hADSCs were significantly intensified by our OPBM strategy. Following OPBM treatment, significant changes were observed in HIF-1α expression, intracellular reactive oxygen species levels, activation of the receptor tyrosine kinase, and glycolytic pathways in hADSCs. In addition, transplantation of OLED-irradiated hADSCs resulted in significantly enhanced limb salvage ratio in a mouse model of hindlimb ischemia. Our OPBM might serve as a new paradigm for stem cell culture systems to develop cell-based therapies in the future.