Utility of Cellular Measurements of Non-Specific Endocytosis to Assess the Target-Independent Clearance of Monoclonal Antibodies.

Past studies have demonstrated higher clearance for monoclonal antibodies possessing increased rates of non-specific endocytosis. However, this metric is oftentimes evaluated indirectly using biophysical techniques or cell surface binding studies that may not provide insight into the specific rates of cellular turnover. Furthermore, few examples evaluating non-specific endocytosis have been reported for a therapeutic antibody that reached clinical assessment. In the current report, we evaluated a therapeutic human immunoglobulin G2 monoclonal antibody targeted against the interleukin-4 receptor alpha chain (IL-4Rα) that exhibited elevated target independent clearance in previous Phase 1 and 2 studies. We confirmed high non-specific clearance of the anti-IL-4Rα antibody as compared to a reference antibody during pharmacokinetic assessments in wild type mice where target-mediated disposition was absent. We then developed a cell-based method capable of measuring cellular protein endocytosis and demonstrated the anti-IL-4Rα antibody exhibited marked non-specific uptake relative to the reference compound. Antibody homology modeling identified the anti-IL-4Rα antibody possessed positive charge patches whose removal via targeted mutations substantially reduced its non-specific endocytosis. We then expanded the scope of the study by evaluating panels of both preclinical and clinically relevant monoclonal antibodies and demonstrate those with the highest rates of non-specific uptake in vitro exhibit elevated target independent clearance, low subcutaneous bioavailability, or both. Our results support the observation that high non-specific endocytosis is a negative attribute in monoclonal antibody development and demonstrate the utility of a generic cell-based screen as a quantitative tool to measure non-specific endocytosis of protein therapeutics at the single-cell level.

Accelerating the in vitro emulation of Alzheimer's disease-associated phenotypes using a novel 3D blood-brain barrier neurosphere co-culture model.

High failure rates in clinical trials for neurodegenerative disorders such as Alzheimer's disease have been linked to an insufficient predictive validity of current animal-based disease models. This has created an increasing demand for alternative, human-based models capable of emulating key pathological phenotypes in vitro. Here, a three-dimensional Alzheimer's disease model was developed using a compartmentalized microfluidic device that combines a self-assembled microvascular network of the human blood-brain barrier with neurospheres derived from Alzheimer's disease-specific neural progenitor cells. To shorten microfluidic co-culture times, neurospheres were pre-differentiated for 21 days to express Alzheimer's disease-specific pathological phenotypes prior to the introduction into the microfluidic device. In agreement with post-mortem studies and Alzheimer's disease in vivo models, after 7 days of co-culture with pre-differentiated Alzheimer's disease-specific neurospheres, the three-dimensional blood-brain barrier network exhibited significant changes in barrier permeability and morphology. Furthermore, vascular networks in co-culture with Alzheimer's disease-specific microtissues displayed localized ß-amyloid deposition. Thus, by interconnecting a microvascular network of the blood-brain barrier with pre-differentiated neurospheres the presented model holds immense potential for replicating key neurovascular phenotypes of neurodegenerative disorders in vitro.

How Organ-on-a-Chip Technology Can Assist in Studying the Role of the Glymphatic System in Neurodegenerative Diseases.

The lack of a conventional lymphatic system that permeates throughout the entire human brain has encouraged the identification and study of alternative clearance routes within the cerebrum. In 2012, the concept of the glymphatic system, a perivascular network that fluidically connects the cerebrospinal fluid to the lymphatic vessels within the meninges via the interstitium, emerged. Although its exact mode of action has not yet been fully characterized, the key underlying processes that govern solute transport and waste clearance have been identified. This review briefly describes the perivascular glial-dependent clearance system and elucidates its fundamental role in neurodegenerative diseases. The current knowledge of the glymphatic system is based almost exclusively on animal-based measurements, but these face certain limitations inherent to in vivo experiments. Recent advances in organ-on-a-chip technology are discussed to demonstrate the technology's ability to provide alternative human-based in vitro research models. Herein, the specific focus is on how current microfluidic-based in vitro models of the neurovascular system and neurodegenerative diseases might be employed to (i) gain a deeper understanding of the role and function of the glymphatic system and (ii) to identify new opportunities for pharmacological intervention.

Empowering engineered muscle in biohybrid pump by extending connexin 43 duration with reduced graphene oxides.

Engineered skeletal muscle act as therapeutics invaluable to treat injured or diseased muscle and a "living" material essential to assemble biological machinery. For normal development, skeletal myoblasts should express connexin 43, one of the gap junction proteins that promote myoblast fusion and myogenesis, during the early differentiation stage. However, myoblasts cultured in vitro often down-regulate connexin 43 before differentiation, limiting myogenesis and muscle contraction. This study demonstrates that tethering myoblasts with reduced graphene oxide (rGO) slows connexin 43 regression during early differentiation and increases myogenic mRNA synthesis. The whole RNA sequencing also confirms that the rGO on cells increases regulator genes for myogenesis, including troponin, while decreasing negative regulator genes. The resulting myotubes generated a three-fold larger contraction force than the rGO-free myotubes. Accordingly, a valveless biohybrid pump assembled with the rGO-tethered muscle increased the fluid velocity and flow rate considerably. The results of this study would provide an important foundation for developing physiologically relevant muscle and powering up biomachines that will be used for various bioscience studies and unexplored applications.

Extracellular Microenvironmental Control for Organoid Assembly.

Organoids, which are multicellular clusters with similar physiological functions to living organs, have gained increasing attention in bioengineering. As organoids become more advanced, methods to form complex structures continue to develop. There is evidence that the extracellular microenvironment can regulate organoid quality. The extracellular microenvironment consists of soluble bioactive molecules, extracellular matrix, and biofluid flow. However, few efforts have been made to discuss the microenvironment optimal to engineer specific organoids. Therefore, this review article examines the extent to which engineered extracellular microenvironments regulate organoid quality. First, we summarize the natural tissue and organ's unique chemical and mechanical properties, guiding researchers to design an extracellular microenvironment used for organoid engineering. Then, we summarize how the microenvironments contribute to the formation and growth of the brain, lung, intestine, liver, retinal, and kidney organoids. The approaches to forming and evaluating the resulting organoids are also discussed in detail. Impact statement Organoids, which are multicellular clusters with similar physiological function to living organs, have been gaining increasing attention in bioengineering. As organoids become more advanced, methods to form complex structures continue to develop. This review article focuses on recent efforts to engineer the extracellular microenvironment in organoid research. We summarized the natural organ's microenvironment, which informs researchers of key factors that can influence organoid formation. Then, we summarize how these microenvironmental controls significantly contribute to the formation and growth of the corresponding brain, lung, intestine, liver, retinal, and kidney organoids. The approaches to forming and evaluating the resulting organoids are discussed in detail, including extracellular matrix choice and properties, culture methods, and the evaluation of the morphology and functionality through imaging and biochemical analysis.

A robust vasculogenic microfluidic model using human immortalized endothelial cells and Thy1 positive fibroblasts.

Human umbilical vein endothelial cells (HUVECs) and stromal cells, such as human lung fibroblasts (FBs), have been widely used to generate functional microvascular networks (µVNs) in vitro. However, primary cells derived from different donors have batch-to-batch variations and limited lifespans when cultured in vitro, which hampers the reproducibility of µVN formation. Here, we immortalize HUVECs and FBs by exogenously expressing human telomerase reverse transcriptase (hTERT) to obtain stable endothelial cell and FB sources for µVN formation in vitro. Interestingly, we find that immortalized HUVECs can only form functional µVNs with immortalized FBs from earlier passages but not from later passages. Mechanistically, we show that Thy1 expression decreases in FBs from later passages. Compared to Thy1 negative FBs, Thy1 positive FBs express higher IGFBP2, IGFBP7, and SPARC, which are important for angiogenesis and lumen formation during vasculogenesis in 3D. Moreover, Thy1 negative FBs physically block microvessel openings, reducing the perfusability of µVNs. Finally, by culturing immortalized FBs on gelatin-coated surfaces in serum-free medium, we are able to maintain the majority of Thy1 positive immortalized FBs to support perfusable µVN formation. Overall, we establish stable cell sources for µVN formation and characterize the functions of Thy1 positive and negative FBs in vasculogenesis in vitro.

Surface tethering of stromal cell-derived factor-1α carriers to stem cells enhances cell homing to ischemic muscle.

Mesenchymal stem cells are promising medicine for treating diseases and tissue defects because of their innate ability to secrete therapeutic factors. Intravenous delivery of stem cells, although favored for its minimal invasiveness, is often plagued by low cellular engraftment in the target tissue. To this end, this study hypothesizes that in situ activation of cellular expression of CXC chemokine 4 (CXCR4) would significantly improve cellular migration to injured tissue. This hypothesis was examined by tethering the surface of stem cells with poly(D,L-lactide-co-glycolide)-block-hyaluronic acid (HA) particles containing stromal cell-derived factor-1α, a model chemokine to sensitize CXCR4. The HA blocks in the particles enhanced the association rate constant to stem cells by 3.3-fold, and in turn, increased the number of cells expressing CXCR4 receptors. Consequently, these cells displayed 1.2-fold higher transendothelial migration in vitro and 1.7-fold greater trafficking to the ischemic hindlimb of a mouse than that of the untethered cells.

Surface Tethering of Inflammation-Modulatory Nanostimulators to Stem Cells for Ischemic Muscle Repair.

Stem cell transplantation has been a promising treatment for peripheral arterial diseases in the past decade. Stem cells act as living bioreactors of paracrine factors that orchestrate tissue regeneration. Prestimulated adipose-derived stem cells (ADSCs) have been proposed as potential candidates but have been met with challenges in activating their secretory activities for clinical use. Here, we propose that tethering the ADSC surface with nanoparticles releasing tumor necrosis factor α (TNFα), named nanostimulator, would stimulate cellular secretory activity in situ. We examined this hypothesis by complexing octadecylamine-grafted hyaluronic acid onto a liposomal carrier of TNFα. Hyaluronic acid increased the liposomal stability and association to CD44 on ADSC surface. ADSCs tethered with these TNFα carriers exhibited up-regulated secretion of proangiogenic vascular endothelial growth factor and immunomodulatory prosteoglandin E2 (PGE2) while decreasing secretion of antiangiogenic pigment epithelium-derived factors. Accordingly, ADSCs tethered with nanostimulators promoted vascularization in a 3D microvascular chip and enhanced recovery of perfusion, walking, and muscle mass in a murine ischemic hindlimb compared to untreated ADSCs. We propose that this surface tethering strategy for in situ stimulation of stem cells would replace the costly and cumbersome preconditioning process and expedite clinical use of stem cells for improved treatments of various injuries and diseases.

Stimulus-Responsive Anti-Oxidizing Drug Crystals and their Ecological Implication.

Various antioxidants are being used to neutralize the harmful effects of reactive oxygen species (ROS) overproduced in diseased tissues and contaminated environments. Polymer-directed crystallization of antioxidants has attracted attention as a way to control drug efficacy through molecular dissolution. However, most recrystallized antioxidants undertake continuous dissolution independent of the ROS level, thus causing side-effects. This study demonstrates a unique method to assemble antioxidant crystals that modulate their dissolution rate in response to the ROS level. We hypothesized that antioxidants recrystallized using a ROS-labile polymer would be triggered to dissolve when the ROS level increases. We examined this hypothesis by using catechin as a model antioxidant. Catechin was recrystallized using polyethylenimine cross-linked with ROS-labile diselanediylbis-(ethane-2,1-diyl)-diacrylate. Catechin crystallized with the ROS-labile polymer displays accelerated dissolution proportional to the H2 O2 concentration. The ROS-responsive catechin crystals protect vascular cells from oxidative insults by activating intracellular glutathione peroxidase expression and, in turn, inhibiting an increase in the intracellular oxidative stress. In addition, ROS-responsive catechin crystals alleviate changes in the heart rate of Daphnia magna in oxidative media. We propose that the results of this study would be broadly useful for improving the therapeutic efficacy of a broad array of drug compounds.

Matrix Topography Regulates Synaptic Transmission at the Neuromuscular Junction.

Recreation of a muscle that can be controlled by the nervous system would provide a major breakthrough for treatments of injury and diseases. However, the underlying basis of how neuron-muscle interfaces are formed is still not understood sufficiently. Here, it is hypothesized that substrate topography regulates neural innervation and synaptic transmission by mediating the cross-talk between neurons and muscles. This hypothesis is examined by differentiating neural stem cells on the myotubes, formed on the substrate with controlled groove width. The substrate with the groove width of 1600 nm, a similar size to the myofibril diameter, serves to produce larger and aligned myotubes than the flat substrate. The myotubes formed on the grooved substrate display increases in the acetylcholine receptor expression. Reciprocally, motor neuron progenitor cells differentiated from neural stem cells innervate the larger and aligned myotubes more actively than randomly oriented myotubes. As a consequence, mature and aligned myotubes respond to glutamate (i.e., an excitatory neurotransmitter) and curare (i.e., a neuromuscular antagonist) more rapidly and homogeneously than randomly oriented myotubes. The results of this study will be broadly useful for improving the quality of engineered muscle used in a series of applications including drug screening, regeneration therapies, and biological machinery assembly.

Surface tethering of stem cells with H2O2-responsive anti-oxidizing colloidal particles for protection against oxidation-induced death.

Mesenchymal stem cells are the new generation of medicine for treating numerous vascular diseases and tissue defects because of their ability to secrete therapeutic factors. Poor cellular survival in an oxidative diseased tissue, however, hinders the therapeutic efficacy. To this end, we hypothesized that tethering the surface of stem cells with colloidal particles capable of discharging antioxidant cargos in response to elevated levels of hydrogen peroxide (H2O2) would maintain survival and therapeutic activity of the stem cells. We examined this hypothesis by encapsulating epigallocatechin gallate (EGCG) and manganese oxide (MnO2) nanocatalysts into particles comprising poly(d,l-lactide-co-glycolide)-block-hyaluronic acid. The MnO2 nanocatalysts catalyzed the decomposition of H2O2 into oxygen gas, which increased the internal pressure of particles and accelerated the release of EGCG by 1.5-fold. Consequently, stem cells exhibited 1.2-fold higher metabolic activity and 2.8-fold higher secretion level of pro-angiogenic factor in sub-lethal H2O2 concentrations. These stem cells, in turn, performed a greater angiogenic potential with doubled number of newly formed mature blood vessels. We envisage that the results of this study will contribute to improving the therapeutic efficacy of a wide array of stem cells.

Long-Term Cryopreservation and Revival of Tissue-Engineered Skeletal Muscle.

IMPACT STATEMENT: The ability to freeze, revive, and prolong the lifetime of tissue-engineered skeletal muscle without incurring any loss of function represents a significant advancement in the field of tissue engineering. Cryopreservation enables the efficient fabrication, storage, and shipment of these tissues. This in turn facilitates multidisciplinary collaboration between research groups, enabling advances in skeletal muscle regenerative medicine, organ-on-a-chip models of disease, drug testing, and soft robotics. Furthermore, the observation that freezing undifferentiated skeletal muscle enhances functional performance may motivate future studies developing stronger and more clinically relevant engineered muscle.

Spatial Organization of Superparamagnetic Iron Oxide Nanoparticles in/on Nano/Microsized Carriers Modulates the Magnetic Resonance Signal.

Superparamagnetic iron oxide nanoparticles (SPIONs) are often encapsulated into drug-carrying nano/microsized particles for simultaneous magnetic resonance (MR) imaging and treatment of diseased tissues. Unfortunately, encapsulated SPIONs may have a limited ability to modulate the T2-weighted relaxation of water protons, but this insight has not been examined systematically. This study demonstrates that SPIONs immobilized on 200 nm diameter poly(lactic- co-glycolic acid) (PLGA) nanoparticles using Pickering emulsification present 18-fold higher relaxivity than encapsulated SPIONs and 1.5-fold higher relaxivity than free SPIONs. In contrast, the SPIONs immobilized on 10 µm diameter PLGA particles exhibit a minor increase in MR relaxivity. This interesting finding will significantly impact current efforts to synthesize and assemble advanced MR contrast agents.

Electrospun Silk Fibroin Nanofibrous Scaffolds with Two-Stage Hydroxyapatite Functionalization for Enhancing the Osteogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells.

The development of functional scaffolds with improved osteogenic potential is important for successful bone formation and mineralization in bone tissue engineering. In this study, we developed a functional electrospun silk fibroin (SF) nanofibrous scaffold functionalized with two-stage hydroxyapatite (HAp) particles, using mussel adhesive-inspired polydopamine (PDA) chemistry. HAp particles were first incorporated into SF scaffolds during the electrospinning process, and then immobilized onto the electrospun SF nanofibrous scaffolds containing HAp via PDA-mediated adhesive chemistry. We obtained two-stage HAp-functionalized SF nanofibrous scaffolds with improved mechanical properties and capable of providing a bone-specific physiological microenvironment. The developed scaffolds were tested for their ability to enhance the osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADMSCs) in vitro and repair bone defect in vivo. To boost their ability for bone repair, we genetically modified hADMSCs with the transcriptional coactivator with PDZ-binding motif (TAZ) via polymer nanoparticle-mediated gene delivery. TAZ is a well-known transcriptional modulator that activates the osteogenic differentiation of mesenchymal stem cells (MSCs). Two-stage HAp-functionalized SF scaffolds significantly promoted the osteogenic differentiation of TAZ-transfected hADMSCs in vitro and enhanced mineralized bone formation in a critical-sized calvarial bone defect model. Our study shows the potential utility of SF scaffolds with nanofibrous structures and enriched inorganic components in bone tissue engineering.

Decellularized Matrix Produced by Mesenchymal Stem Cells Modulates Growth and Metabolic Activity of Hepatic Cell Cluster.

Miniature organlike three-dimensional cell clusters often called organoids have emerged as a useful tool for both fundamental and applied bioscience studies. However, there is still a great need to improve the quality of organoids to a level where they exhibit similar biological functionality to an organ. To this end, we hypothesized that a decellularized matrix derived from mesenchymal stem cell (MSC) could regulate the phenotypic and metabolic activity of organoids. This hypothesis was examined by culturing cells of interest in the decellularized matrix of MSCs cultured on a 2D substrate at confluency or in the form of spheroids. The decellularized matrix prepared with MSC spheroids showed a 3D porous structure with a higher content of extracellular matrix molecules than the decellularized matrix derived from MSCs cultured on a 2D substrate. HepG2 hepatocarcinoma cells, which retain the metabolic activity of hepatocytes, were cultured in these decellularized matrices. Interestingly, the decellularized matrix from the MSC spheroids served to develop the hepatic cell clusters with higher levels of E-cadherin-mediated cell-cell adhesion and detoxification activity than the decellularized matrix from the MSCs cultured on a 2D substrate. Overall, the results of this study are useful in improving biological functionality of a wide array of organoids.

Investigating the Life Expectancy and Proteolytic Degradation of Engineered Skeletal Muscle Biological Machines.

A combination of techniques from 3D printing, tissue engineering and biomaterials has yielded a new class of engineered biological robots that could be reliably controlled via applied signals. These machines are powered by a muscle strip composed of differentiated skeletal myofibers in a matrix of natural proteins, including fibrin, that provide physical support and cues to the cells as an engineered basement membrane. However, maintaining consistent results becomes challenging when sustaining a living system in vitro. Skeletal muscle must be preserved in a differentiated state and the system is subject to degradation by proteolytic enzymes that can break down its mechanical integrity. Here we examine the life expectancy, breakdown, and device failure of engineered skeletal muscle bio-bots as a result of degradation by three classes of proteases: plasmin, cathepsin L, and matrix metalloproteinases (MMP-2 and MMP-9). We also demonstrate the use of gelatin zymography to determine the effects of differentiation and inhibitor concentration on protease expression. With this knowledge, we are poised to design the next generation of complex biological machines with controllable function, specific life expectancy and greater consistency. These results could also prove useful for the study of disease-specific models, treatments of myopathies, and other tissue engineering applications.

Balanced Effects of Surface Reactivity and Self-Association of Bifunctional Polyaspartamide on Stem Cell Adhesion.

Extensive efforts have been made to regulate surface wettability using bivalent polymers composed of hydrophobic surface-reactive groups and hydrophilic groups. To further enhance the controllability, this study demonstrates that the balance between the surface reactivity and self-aggregation of bivalent poly(hydroxyethyl-co-methacryloxyethyl aspartamide) (PHMAA) is crucial in controlling the wettability of methacrylated glass and thus the adhesion of stem cells. In particular, the wettability of the glass and the subsequent cell spreading became maximal with PHMAA that led to the largest and most uniform coverage of hydroxyl groups. In summary, this study would be useful in advancing various molecules used for surface engineering.

Nanostructured Tendon-Derived Scaffolds for Enhanced Bone Regeneration by Human Adipose-Derived Stem Cells.

Decellularized matrix-based scaffolds can induce enhanced tissue regeneration due to their biochemical, biophysical, and mechanical similarity to native tissues. In this study, we report a nanostructured decellularized tendon scaffold with aligned, nanofibrous structures to enhance osteogenic differentiation and in vivo bone formation of human adipose-derived stem cells (hADSCs). Using a bioskiving method, we prepared decellularized tendon scaffolds from tissue slices of bovine Achilles and neck tendons with or without fixation, and investigated the effects on physical and mechanical properties of decellularized tendon scaffolds, based on the types and concentrations of cross-linking agents. In general, we found that decellularized tendon scaffolds without fixative treatments were more effective in inducing osteogenic differentiation and mineralization of hADSCs in vitro. When non-cross-linked decellularized tendon scaffolds were applied together with hydroxyapatite for hADSC transplantation in critical-sized bone defects, they promoted bone-specific collagen deposition and mineralized bone formation 4 and 8 weeks after hADSC transplantation, compared to conventional collagen type I scaffolds. Interestingly, stacking of decellularized tendon scaffolds cultured with osteogenically committed hADSCs and those containing human cord blood-derived endothelial progenitor cells (hEPCs) induced vascularized bone regeneration in the defects 8 weeks after transplantation. Our study suggests that biomimetic nanostructured scaffolds made of decellularized tissue matrices can serve as functional tissue-engineering scaffolds for enhanced osteogenesis of stem cells.

Rupture force of cell adhesion ligand tethers modulates biological activities of a cell-laden hydrogel.

Recent efforts to design a synthetic extracellular matrix for cell culture, engineering, and therapies greatly contributed to addressing biological roles of types and spatial organization of cell adhesion ligands. It is often suggested that ligand-matrix bond strength is another path to regulate cell adhesion and activities; however tools are lacking. To this end, this study demonstrates that a hydrogel coupled with integrin-binding deoxyribonucleic acid (DNA) tethers with pre-defined rupture forces can modulate cell adhesion, differentiation, and secretion activities due to the changes in the number and, likely, force of cells adhered to a gel. The rupture force of DNA tethers was tuned by altering the spatial arrangement of matrix-binding biotin groups. The DNA tethers were immobilized on a hydrogel of alginate grafted with biotin using avidin. Mesenchymal stem cells showed enhanced adhesion, neural differentiation, and paracrine secretion when cultured on the gel coupled with DNA tethers with higher rupture forces. Such innovative cell-matrix interface engineering would be broadly useful for a series of materials used for fundamental and applied studies on biological cells.

Alginate Sulfates Mitigate Binding Kinetics of Proangiogenic Growth Factors with Receptors toward Revascularization.

Ever since proangiogenic growth factors have been used as a vascular medicine to treat tissue ischemia, efforts have been increasingly made to develop a method to enhance efficacy of growth factors in recreating microvascular networks, especially at low dose. To this end, we hypothesized that polysaccharides substituted with sulfate groups would amplify growth factor receptor activation and stimulate phenotypic activities of endothelial cells involved in neovascularization. We examined this hypothesis by modifying alginate with a controlled number of sulfates and using it to derive a complex with vascular endothelial growth factor (VEGF), as confirmed with fluorescence resonance energy transfer (FRET) assay. Compared with the bare VEGF and with a mixture of VEGF and unmodified alginates, the VEGF complexed with alginate sulfates significantly reduced the dissociation rate with the VEGFR-2, elevated VEGFR-2 phosphorylation level, and increased the number of endothelial sprouts in vitro. Furthermore, the VEGF-alginate sulfate complex improved recovery of perfusion in an ischemic hindlimb of a mouse due to the increase of the capillary density. Overall, this study not only demonstrates an important cofactor of VEGF but also uncovers an underlying mechanism by which the cofactor mitigates the VEGF-induced signaling involved in the binding kinetics and activation of VEGFR. We therefore believe that the results of this study will be highly useful in improving the therapeutic efficacy of various growth factors and expediting their uses in clinical treatments of wounds and tissue defects.