Cellular Layer-by-Layer Coculture Platform Using Biodegradable, Nanoarchitectured Membranes for Stem Cell Therapy

Stem cells are regulated <i>in vivo</i> through interactions with the surrounding microenvironments in a three-dimensional (3D) manner. A coculture of stem cells with desired cell types, which recapitulates the complex <i>in vivo</i> cell–cell communications, has been reported as an effective method to direct stem cell differentiation into specific lineage. However, conventional bilayer coculture systems employ membranes of microscale thickness and low porosity, which limit interaction between cocultured cells for efficient stem cell differentiation. Furthermore, conventional coculture systems require cell-impairing enzyme treatment to harvest the cells from the membranes. Here, we developed a cellular layer-by-layer (cLbL) coculture platform using biodegradable, nanothin, highly porous (BNTHP) membranes. Equipped with more porous and thinner membranes, the cLbL coculture platform better mimicked the <i>in vivo</i> 3D microenvironment and promoted cellular cross-talks between cocultured cells which occurred in nanoscale, resulting in more efficient stem cell differentiation compared to the conventional bilayer coculture systems. Furthermore, biodegradibility, biocompatibility, and highly flexibility of BNTHP membranes enabled conversion of the cell-attached membranes into implantable 3D cell constructs, thus avoiding harmful enzymatic harvesting of the cells. The cLbL platform may be an effective method to induce stem cell differentiation and facilitate cell implantation for stem cell therapy

Gold Nanoparticle/Graphene Oxide Hybrid Sheets Attached on Mesenchymal Stem Cells for Effective Photothermal Cancer Therapy

Cell-mediated nanoparticle delivery has been proposed for an effective cancer therapy. However, there are limitations in loading nanoparticles within cells as the internalized nanoparticles cause cytotoxicity and leak out of the cells via exocytosis. Here, we introduce hybrid sheets composed of gold nanoparticles (AuNPs) and graphene oxide (GO), which stably adhere to the cell surface and exhibit a remarkable photothermal effect. To form AuNP/GO sheets in which GO is sandwiched between two AuNP monolayers, AuNPs are coated with α-synuclein protein and subsequently adsorbed onto GO sheets. Attaching AuNP/GO sheets to the tumor-tropic mesenchymal stem cell (MSC) surface enhances the loading efficiency of AuNPs in MSCs by avoiding the cytotoxicity and exocytosis issues. Furthermore, the tight packing of AuNPs on microscaled GO sheets enhances the photothermal effect via strong plasmon coupling between AuNPs. The injection of AuNP/GO sheet-attached MSCs into tumor-bearing mice significantly improves the photothermal therapeutic efficacy by delivering larger amounts of AuNPs to the tumor and generating higher heat at the tumor region compared to injection of AuNP-internalized MSCs. The system of attaching AuNP/GO hybrid sheets to the tumor-tropic cell surface may be an effective platform for cancer therapy

Cooperative Catechol-Functionalized Polypept(o)ide Brushes and Ag Nanoparticles for Combination of Protein Resistance and Antimicrobial Activity on Metal Oxide Surfaces

Prevention of biofouling and microbial contamination of implanted biomedical devices is essential to maintain their functionality and biocompatibility. For this purpose, polypept­(o)­ide block copolymers have been developed, in which a protein-resistant polysarcosine (pSar) block is combined with a dopamine-modified poly­(glutamic acid) block for surface coating and silver nanoparticles (Ag NPs) formation. In the development of a novel, versatile, and biocompatible antibacterial surface coating, block lengths pSar were varied to derive structure–property relationships. Notably, the catechol moiety performs two important tasks in parallel; primarily it acts as an efficient anchoring group to metal oxide surfaces, while it furthermore induces the formation of Ag NPs. Attributing to the dual function of catechol moieties, antifouling pSar brush and antimicrobial Ag NPs can not only adhere stably on metal oxide surfaces, but also display passive antifouling and active antimicrobial activity, showing good biocompatibility simultaneously. The developed strategy seems to provide a promising platform for functional modification of biomaterials surface to preserve their performance while reducing the risk of bacterial infections

Nanothin Coculture Membranes with Tunable Pore Architecture and Thermoresponsive Functionality for Transfer-Printable Stem Cell-Derived Cardiac Sheets

Coculturing stem cells with the desired cell type is an effective method to promote the differentiation of stem cells. The features of the membrane used for coculturing are crucial to achieving the best outcome. Not only should the membrane act as a physical barrier that prevents the mixing of the cocultured cell populations, but it should also allow effective interactions between the cells. Unfortunately, conventional membranes used for coculture do not sufficiently meet these requirements. In addition, cell harvesting using proteolytic enzymes following coculture impairs cell viability and the extracellular matrix (ECM) produced by the cultured cells. To overcome these limitations, we developed nanothin and highly porous (NTHP) membranes, which are ∼20-fold thinner and ∼25-fold more porous than the conventional coculture membranes. The tunable pore size of NTHP membranes at the nanoscale level was found crucial for the formation of direct gap junctions-mediated contacts between the cocultured cells. Differentiation of the cocultured stem cells was dramatically enhanced with the pore size-customized NTHP membrane system compared to conventional coculture methods. This was likely due to effective physical contacts between the cocultured cells and the fast diffusion of bioactive molecules across the membrane. Also, the thermoresponsive functionality of the NTHP membranes enabled the efficient generation of homogeneous, ECM-preserved, highly viable, and transfer-printable sheets of cardiomyogenically differentiated cells. The coculture platform developed in this study would be effective for producing various types of therapeutic multilayered cell sheets that can be differentiated from stem cells

Reversible Cell Layering for Heterogeneous Cell Assembly Mediated by Ionic Cross-Linking of Chitosan and a Functionalized Cell Surface Membrane

Current heterogeneous cell assembly techniques in coculture systems rely on irreversible cell layering or a cell separation membrane. However, the techniques possess major drawbacks of inefficiency in direct interactions of the assembled cell layers and cell separation following coculture, which hamper characterization and therapeutic applications of the cells following coculture. Here, we develop a reversible cell layering platform for assembly of heterogeneous cells that allows both active direct cell–cell interactions and facile cell separation. Anionic maleimide-chondroitin-sulfate is grafted onto the surface membrane of myogenic C2C12 cells and human mesenchymal stem cells (hMSCs) to modify the surface charge of the cells without cytotoxicity. A highly porous chitosan thin film is formed <i>in situ</i> interspacing between the heterogeneous cell layers via ionic cross-linking of cationic chitosan and anionic functionalized cells, forming compactly assembled double-layered cell constructs. The chitosan film enables layering of the cells, which allows active direct interactions between the cell layers, and facile delayering of the cells through simple treatment with mild shear stress. The developed platform promotes the myogenic commitment of hMSCs via direct contact with C2C12 cells, mimicking the interactions that trigger stem cell differentiation <i>in vivo</i>. Delivery of the myogenic committed cells to muscle-injured animal models shows evident muscle regeneration

Dual Roles of Graphene Oxide To Attenuate Inflammation and Elicit Timely Polarization of Macrophage Phenotypes for Cardiac Repair

Development of localized inflammatory environments by M1 macrophages in the cardiac infarction region exacerbates heart failure after myocardial infarction (MI). Therefore, the regulation of inflammation by M1 macrophages and their timely polarization toward regenerative M2 macrophages suggest an immunotherapy. Particularly, controlling cellular generation of reactive oxygen species (ROS), which cause M1 differentiation, and developing M2 macrophage phenotypes in macrophages propose a therapeutic approach. Previously, stem or dendritic cells were used in MI for their anti-inflammatory and cardioprotective potentials and showed inflammation modulation and M2 macrophage progression for cardiac repair. However, cell-based therapeutics are limited due to invasive cell isolation, time-consuming cell expansion, labor-intensive and costly <i>ex vivo</i> cell manipulation, and low grafting efficiency. Here, we report that graphene oxide (GO) can serve as an antioxidant and attenuate inflammation and inflammatory polarization of macrophages <i>via</i> reduction in intracellular ROS. In addition, GO functions as a carrier for interleukin-4 plasmid DNA (IL-4 pDNA) that propagates M2 macrophages. We synthesized a macrophage-targeting/polarizing GO complex (MGC) and demonstrated that MGC decreased ROS in immune-stimulated macrophages. Furthermore, DNA-functionalized MGC (MGC/IL-4 pDNA) polarized M1 to M2 macrophages and enhanced the secretion of cardiac repair-favorable cytokines. Accordingly, injection of MGC/IL-4 pDNA into mouse MI models attenuated inflammation, elicited early polarization toward M2 macrophages, mitigated fibrosis, and improved heart function. Taken together, the present study highlights a biological application of GO in timely modulation of the immune environment in MI for cardiac repair. Current therapy using off-the-shelf material GO may overcome the shortcomings of cell therapies for MI