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Messenger RNA (mRNA) delivery provides gene therapy with the potential to achieve transient therapeutic efficacy without risk of insertional mutagenesis. Amongst other applications, mRNA can be employed as a platform to deliver gene editing molecules, to achieve protein expression as an alternative to enzyme replacement therapies, and to express chimeric antigen receptors (CARs) on immune cells for the treatment of cancer. We designed a novel microfluidic device that allows for efficient mRNA delivery via volume exchange for convective transfection (VECT). In the device, cells flow through a ridged channel that enforces a series of ultra-fast and large intensity deformations able to transiently open pores and induce convective transport of mRNA into the cell. Here, we describe efficient delivery of mRNA into T cells, natural killer (NK) cells and hematopoietic stem and progenitor cells (HSPCs), three human primary cell types widely used for ex vivo gene therapy applications. Results demonstrate that the device can operate at a wide range of cell and payload concentrations and that ultra-fast compressions do not have a negative impact on T cell function, making this a novel and competitive platform for the development of ex vivo mRNA-based gene therapies and other cell products engineered with mRNA.

Paracrine signals produced from stem cells influence tissue regeneration by inducing the differentiation of endogenous stem or progenitor cells. However, many recent studies that have investigated paracrine signaling of stem cells have relied on either two-dimensional transwell systems or conditioned medium culture, neither of which provide optimal culture microenvironments for elucidating the effects of paracrine signals in vivo. In this study, we recapitulated in vivo-like paracrine signaling of human mesenchymal stem cells (hMSCs) to enhance functional neuronal differentiation of human neural stem cells (hNSCs) in three-dimensional (3D) extracellular matrices (ECMs) within a microfluidic array platform. In order to amplify paracrine signaling, hMSCs were genetically engineered using cationic polymer nanoparticles to overexpress glial cell-derived neurotrophic factor (GDNF). hNSCs were cultured in 3D ECM hydrogel used to fill central channels of the microfluidic device, while GDNF-overexpressing hMSCs (GDNF-hMSCs) were cultured in channels located on both sides of the central channel. This setup allowed for mimicking of paracrine signaling between genetically engineered hMSCs and endogenous hNSCs in the brain. Co-culture of hNSCs with GDNF-hMSCs in the 3D microfluidic system yielded reduced glial differentiation of hNSCs while significantly enhancing differentiation into neuronal cells including dopaminergic neurons. Neuronal cells produced from hNSCs differentiating in the presence of GDNF-hMSCs exhibited functional neuron-like electrophysiological features. The enhanced paracrine ability of GDNF-hMSCs was finally confirmed using an animal model of hypoxic-ischemic brain injury. This study demonstrates the presented 3D microfluidic array device can provide an efficient co-culture platform and provide an environment for paracrine signals from transplanted stem cells to control endogenous neuronal behaviors in vivo.

Engineering physiologically-relevant in vitro models of human organs remains a fundamental challenge. Despite significant strides made within the field, many promising organ-on-a-chip models fall short in recapitulating cellular interactions with neighboring cell types, surrounding extracellular matrix (ECM), and exposure to soluble cues due, in part, to the formation of artificial structures that obstruct >50% of the surface area of the ECM. Here, we report a three-dimensional (3D) cell culture platform based upon hydrophobic patterning of hydrogels that is capable of precisely generating a 3D ECM within a microfluidic channel with an interaction area >95%. In this study, for demonstrative purposes, type I collagen (COL1), Matrigel (MAT), COL1/MAT mixture, hyaluronic acid (HA), and cell-laden MAT were formed in the device. We demonstrate three potential applications, including creating a 3D endothelium model, studying the interstitial migration of cancer cells, and analyzing stem cell differentiation in a 3D environment. Our hydrophobic patterned-based 3D cell culture device provides the ease-of-fabrication and flexibility necessary for broad potential applications in organ-on-a-chip platforms.

Plasticity and reciprocity of breast cancer cells to various extracellular matrice (ECMs) are three-dimensionally analyzed in quantitative way in a novel and powerful microfluidic in vitro platform. This successfully demonstrates the metastatic potential of cancer cells and their effective strategies of ECM proteolytic remodeling and morphological change, while interacting with other cells and invading into heterogeneous ECMs., International Research & Development Program (Grant 2009-00631), National Research Foundation of Korea (Basic Science Research Program Grant 2012-022481)

The construction of well-controllable in vitro models of physiological and pathological vascular endothelium remains a fundamental challenge in tissue engineering and drug development. Here, we present an approach for forming a synthetic endothelial extracellular matrix (ECM) that closely resembles that of the native structure by locally depositing basement membrane materials onto type 1 collagen nanofibers only in a region adjacent to the endothelial cell (EC) monolayer. Culturing the EC monolayer on this synthetic endothelial ECM remarkably enhanced its physiological properties, reducing its vascular permeability, and promoting a stabilized, quiescent phenotype. We demonstrated that the EC monolayer on the synthetic endothelial ECM neither creates non-physiological barriers to cell-cell or cell-ECM interactions, nor hinders molecular diffusion of growth factors and other molecules. The synthetic endothelial ECM and vascular endothelium on it may help us enter in a new phase of research in which various models of the biological barrier behavior can be tested experimentally., Korea (South). Ministry of Science, ICT and Future Planning (National Research Foundation of Korea. Pioneer Research Center Program (NRF-2015M3C1A3002152), Korea (South). Ministry of Science, ICT and Future Planning (BioNano Health-Guard Research Center Global Frontier Project H-GUARD_2014M3A6B2060524)

Aptamers have recently emerged as reliable and promising targeting agents in the field of biology. However, their therapeutic potential has yet to be completely assessed due to their poor pharmacokinetics for systemic administration. Here, we describe a novel aptamer-antibody complex, designated an "oligobody" (oligomer+antibody) that may overcome the therapeutic limitations of aptamers. To provide proof-of-principle study, we investigated the druggability of oligobody in vivo using cotinine conjugated t44-OMe aptamer, which is specific for the sequence of pegaptanib, and an anti-cotinine antibody. The antibody part of oligobody resulted in extended in vivo pharmacokinetics of the aptamer without influencing its binding affinity. Moreover, the aptamer of oligobody penetrated deeply into the tumor tissues whereas the anti-VEGF antibody did not. Finally, the systemic administration of this oligobody reduced the tumor burden in a xenograft mouse model. Together, these results suggested that our oligobody strategy may represent a novel platform for rapid, low-cost and high-throughput cancer therapy.

Here, a growth-factor-integrated natural extracellular matrix of type I collagen is presented that induces angiogenesis. The developed matrix adapts type I collagen nanofibers integrated with synthetic colloidal particles of recombinant bacteriophages that display vascular endothelial growth factor (VEGF). The integration is achieved during or after gelation of the type I collagen and the matrix enables spatial delivery of VEGF into a desired region. Endothelial cells that contact the VEGF are found to invade into the matrix to form tube-like structures both in vitro and in vivo, proving the angiogenic potential of the matrix.

We report a microfluidic array for investigating and quantitatively analyzing human neural stem cell (hNSC) self-renewal and differentiation in an in vivo-like microenvironment. NSC niche conditions, including three-dimensional (3D) extracellular matrices and low oxygen tension, were effectively reconstituted in the microfluidic array in a combinatorial manner. The array device was fabricated to be detachable, rendering it compatible with quantitative real-time polymerase chain reaction for quantifying the effects of the biomimetic conditions on hNSC self-renewal and differentiation. We show that throughput of 3D cell culture and quantitative analysis can be increased. We also show that 3D hypoxic microenvironments maintain hNSC self-renewal capacity and direct neuronal commitment during hNSC differentiation.

Here, we report a unique method to quantify the effects of in vivo-like extracellular matrix (ECM) for guiding differentiation of neural stem cells (NSCs) in three-dimensional (3D) microenvironments using quantitative real-time polymerase chain reaction (qRT-PCR). We successfully monitored and quantified differentiation of NSCs in small volume ECMs and found that differentiation of NSCs, especially those differentiating towards neuronal and oligodendrocytic lineages, is significantly enhanced by 3D microenvironments reconstituted in the microfluidic channels.

Microfluidic cell culture assays are versatile tools for studying cell migration, particularly angiogenesis. Such assays can deliver precisely controlled linear gradients of chemical stimuli to cultured cells in a microfluidic channel, offering excellent optical resolution and in situ monitoring of cellular morphogenesis in response to a gradient. Microfluidic cell culture assays provide a chemical gradient subject to molecular diffusion, although cellular metabolism can perturb it. The actual gradient perturbed by cells has not been precisely described in the context of regulated cellular morphogenesis. We modeled the chemical gradient in a microfluidic channel by simulating the analyte(VEGF) distribution during cellular interactions. The results were experimentally verified by monitoring sprouting angiogenic response from a monolayer of human umbilical vein endothelial cells (hUVECs) into a type 1 collagen scaffold. The simulation provided a basis for understanding a real distribution of the analyte interrupted by cells in microfluidic device. The new protocol enables one to quantify the morphogenesis of hUVECs under a flat, less-steep, or steep gradient.

Spatiotemporal analysis of the inflammatory response has been limited by the difficulties of in vivo imaging and reconstitution of inflammation in vitro. Here, we present a novel method for establishing in vivo-like inflammatory models in a microfluidic device and quantitatively measuring the three-dimensional transmigration of neutrophils during the inflammatory process. This enabled us to concurrently characterize transendothelial migration behaviors of neutrophils under the influence of various inflammatory stimuli.

Sprouting angiogenesis requires a coordinated guidance from a variety of angiogenic factors. Here, we have developed a unique hydrogel incorporating microfluidic platform which mimics the physiological microenvironment in 3D under a precisely orchestrated gradient of soluble angiogenic factors, VEGF and ANG-1. The system enables the quantified investigation in chemotactic response of endothelial cells during the collective angiogenic sprouting process. While the presence of a VEGF gradient alone was sufficient in inducing a greater number of tip cells, addition of ANG-1 to the VEGF gradient enhanced the number of tip cells that are attached to collectively migrated stalk cells. The chemotactic response of tip cells attracted by the VEGF gradient and the stabilizing role of ANG-1 were morphologically investigated, elucidating the 3D co-operative migration of tip and stalk cells as well as their structures. We found that ANG-1 enhanced the connection of the stalk cells with the tip cells, and then the direct connection regulated the morphogenesis and/or life cycle of stalk cells.