Aspiration-mediated hydrogel micropatterning using rail-based open microfluidic devices for high-throughput 3D cell culture.

Microfluidics offers promising methods for aligning cells in physiologically relevant configurations to recapitulate human organ functionality. Specifically, microstructures within microfluidic devices facilitate 3D cell culture by guiding hydrogel precursors containing cells. Conventional approaches utilize capillary forces of hydrogel precursors to guide fluid flow into desired areas of high wettability. These methods, however, require complicated fabrication processes and subtle loading protocols, thus limiting device throughput and experimental yield. Here, we present a swift and robust hydrogel patterning technique for 3D cell culture, where preloaded hydrogel solution in a microfluidic device is aspirated while only leaving a portion of the solution in desired channels. The device is designed such that differing critical capillary pressure conditions are established over the interfaces of the loaded hydrogel solution, which leads to controlled removal of the solution during aspiration. A proposed theoretical model of capillary pressure conditions provides physical insights to inform generalized design rules for device structures. We demonstrate formation of multiple, discontinuous hollow channels with a single aspiration. Then we test vasculogenic capacity of various cell types using a microfluidic device obtained by our technique to illustrate its capabilities as a viable micro-manufacturing scheme for high-throughput cellular co-culture.

High-throughput injection molded microfluidic device for single-cell analysis of spatiotemporal dynamics.

Single-cell level analysis of various cellular behaviors has been aided by recent developments in microfluidic technology. Polydimethylsiloxane (PDMS)-based microfluidic devices have been widely used to elucidate cell differentiation and migration under spatiotemporal stimulation. However, microfluidic devices fabricated with PDMS have inherent limitations due to material issues and non-scalable fabrication process. In this study, we designed and fabricated an injection molded microfluidic device that enables real-time chemical profile control. This device is made of polystyrene (PS), engineered with channel dimensions optimized for injection molding to achieve functionality and compatibility with single cell observation. We demonstrated the spatiotemporal dynamics in the device with computational simulation and experiments. In temporal dynamics, we observed extracellular signal-regulated kinase (ERK) activation of PC12 cells by stimulating the cells with growth factors (GFs). Also, we confirmed yes-associated protein (YAP) phase separation of HEK293 cells under stimulation using sorbitol. In spatial dynamics, we observed the migration of NIH 3T3 cells (transfected with Lifeact-GFP) under different spatiotemporal stimulations of PDGF. Using the injection molded plastic devices, we obtained comprehensive data more easily than before while using less time compared to previous PDMS models. This easy-to-use plastic microfluidic device promises to open a new approach for investigating the mechanisms of cell behavior at the single-cell level.

Tumor spheroid-on-a-chip: a standardized microfluidic culture platform for investigating tumor angiogenesis.

The field of microfluidics-based three-dimensional (3D) cell culture system is rapidly progressing from academic proof-of-concept studies to valid solutions to real-world problems. Polydimethylsiloxane (PDMS)-based platform has been widely adopted as in vitro platforms for mimicking tumor microenvironment. However, PDMS has not been welcomed as a standardized commercial application for preclinical screening due to inherent material limitations that make it difficult to scale-up production. Here, we present an injection-molded plastic array 3D spheroid culture platform (Sphero-IMPACT). The platform is made of polystyrene (PS) in a standardized 96-well plate format with a user-friendly interface. This interface describes a simpler design that incorporates a tapered hole in the center of the rail to pattern a large spheroid with 3D extracellular matrix and various cell types. This hole is designed to accommodate standard pipette tip for automated system. The platform that mediate open microfluidics allows implement spontaneous fluid patterning with high repeatability from the end user. To demonstrate versatile use of the platform, we developed 3D perfusable blood vessel network and tumor spheroid assays. In addition, we established a tumor spheroid induced angiogenesis model that can be applicable for drug screening. Sphero-IMPACT has the potential to provide a robust and reproducible in vitro assay related to vascularized cancer research. This easy-to-use, ready-to-use platform can be translated into an enhanced preclinical model that faithfully reflects the complex tumor microenvironment.

Human Ocular Angiogenesis-Inspired Vascular Models on an Injection-Molded Microfluidic Chip.

Angiogenic sprouting, which is the growth of new blood vessels from pre-existing vessels, is orchestrated by cues from the cellular microenvironment, such as spatially controlled gradients of angiogenic factors. However, current in vitro models are less scalable for in-depth studies of angiogenesis. In this study, a plastic-based microfluidic chip is developed to reconstruct in vitro 3D vascular networks. The main disadvantages of the preexisting system are identified, namely, the low productivity and difficulty of experiments, and a breakthrough is suggested while minimizing disadvantages. The selection of plastic materials contributes to the productivity and usability of in vitro devices. By adopting this material, this chip offers simple fluid patterning, facilitating the construction of a cell-culture microenvironment. Compared with previous systems, the chip, which can form both inward and outwardly radial vascular sprouting, demonstrates the growth of functional, morphologically integral microvessels. The developed angiogenic model yields dose-dependent results for antiangiogenic drug screening. This model may contribute significantly not only to vascular studies under normal and pathological conditions, but also to fundamental research on the ocular neovascularization. Furthermore, it can be applied as a tool for more practical, extended preclinical research, providing an alternative to animal experiments.

High-Throughput Microfluidic 3D Cytotoxicity Assay for Cancer Immunotherapy (CACI-IMPACT Platform).

Adoptive cell transfer against solid tumors faces challenges to overcome tumor microenvironment (TME), which plays as a physical barrier and provides immuno-suppressive conditions. Classical cytotoxicity assays are widely used to measure killing ability of the engineered cytotoxic lymphocytes as therapeutics, but the results cannot represent the performance in clinical application due to the absence of the TME. This paper describes a 3D cytotoxicity assay using an injection molded plastic array culture (CACI-IMPACT) device for 3D cytotoxicity assay to assess killing abilities of cytotoxic lymphocytes in 3D microenvironment through a spatiotemporal analysis of the lymphocytes and cancer cells embedded in 3D extra cellular matrix (ECM). Rail-based microfluidic design was integrated within a single 96-well and the wells were rectangularly arrayed in 2 × 6 to enhance the experimental throughput. The rail-based microstructures facilitate hydrogel patterning with simple pipetting so that hydrogel pre-solution aspirated with 10 µl pipette can be patterned in 10 wells within 30 s. To demonstrate 3D cytotoxicity assay, we patterned HeLa cells encapsulated by collagen gel and observed infiltration, migration and cytotoxic activity of NK-92 cells against HeLa cells in the collagen matrix. We found that 3D ECM significantly reduced migration of cytotoxic lymphocytes and access to cancer cells, resulting in lower cytotoxicity compared with 2D assays. In dense ECM, the physical barrier function of the 3D matrix was enhanced, but the cytotoxic lymphocytes effectively killed cancer cells once they contacted with cancer cells. The results implied ECM significantly influences migration and cytotoxicity of cytotoxic lymphocytes. Hence, the CACI-IMPACT platform, enabling high-throughput 3D co-culture of cytotoxic lymphocyte with cancer cells, has the potential to be used for pre-clinical evaluation of cytotoxic lymphocytes engineered for immunotherapy against solid tumors.

Engineering tumor vasculature on an injection-molded plastic array 3D culture (IMPACT) platform.

Recent advances in microfluidic organ-on-a-chip technology have enabled the growth of 3D microphysiological systems for diverse biological studies. Fabrication and usage limitations inherent to conventional soft lithographic polydimethylsiloxane (PDMS) based microfluidic platforms drive demands for more accessible, standardized, and mass producible platforms for wider applications. Here, we introduce a novel injection-molded plastic array 3D culture (IMPACT) platform, a microfluidic system designed for easy and diverse patterning of 3D cellular hydrogel. The flexibility of the IMPACT platform enabled simultaneous high-content morphological profiling of the effect of nine different types of tumor cells on vascular formation. Moreover, screening of three different known anti-tumor drugs (5-FU, axitinib and cetuximab) was done at various delivered dosages. We observed distinct and expected molecular mechanism dependent response on both tumor and vasculature in response to treatment, confirming the applicability of the IMPACT as high-content drug testing tool. Therefore, we propose IMPACT as the next generation of 3D microfluidic co-culture platform compatible with any biological, clinical, and pharmaceutical investigations requiring robust high-throughput and high-content assays.

Modeling neural circuit, blood-brain barrier, and myelination on a microfluidic 96 well plate.

Microfluidics have enabled a wide range of experimental possibilities in the field of neuroscience. Unfortunately, the wider scale adoption of polydimethylsiloxane (PDMS) based microfluidic devices faces challenges due to inherent material compatibility issues and lack of standardized manufacturable devices. In this work, we present an injection molded plastic array three-dimensional (3D) neuron culture platform (Neuro-IMPACT) made of polystyrene (PS) with a standard 96-well plate form factor that can recapitulate elements of both the central and peripheral nervous systems. A standardized in vitro platform for neuron culture will facilitate the development of new therapies for neurodegenerative diseases, as they would enable quantitative analysis based on imaging as well as biochemical analysis. To demonstrate the versatility of Neuro-IMPACT, we modeled physiologically relevant complex co-culture models such as a 3D neuronal network, blood-brain barrier, and myelination. The Neuro-IMPACT offers a high-throughput screening compatible platform with the ability to engineer the neuronal microenvironment to aid both basic and applied neuroscience research.

Microfluidic-based vascularized microphysiological systems.

Microphysiological systems have emerged in the last decade to provide an alternative to in vivo models in basic science and pharmaceutical research. In the field of vascular biology, in particular, there has been a lack of a suitable in vitro model exhibiting a three-dimensional structure and the physiological function of vasculature integrated with organ-on-a-chip models. The rapid development of organ-on-a-chip technology is well positioned to fulfill unmet needs. Recently, functional integration of vasculature with diverse microphysiological systems has been increasing. This recent trend corresponds to emerging research interest in how the vascular system contributes to various physiological and pathological conditions. This innovative platform has undergone significant development, but adoption of this technology by end-users and researchers in biology is still a work in progress. Therefore, it is critical to focus on simplification and standardization to promote the distribution and acceptance of this technology by the end-users. In this review, we will introduce the latest developments in vascularized microphysiological systems and summarize their outlook in basic research and drug screening applications.

Microfluidics within a well: an injection-molded plastic array 3D culture platform.

Polydimethylsiloxane (PDMS) has been widely used in fabricating microfluidic devices for prototyping and proof-of-concept experiments. Due to several material limitations, PDMS has not been widely adopted for commercial applications that require large-scale production. This paper describes a novel injection-molded plastic array 3D culture (IMPACT) platform that incorporates a microfluidic design to integrate patterned 3D cell cultures within a single 96-well (diameter = 9 mm) plate. Cell containing gels can be sequentially patterned by capillary-guided flow along the corner and narrow gaps designed within the 96-well form factor. Compared to PDMS-based hydrophobic burst valve designs, this work utilizes hydrophilic liquid guides to obtain rapid and reproducible patterned gels for co-cultures. When a liquid droplet (i.e. cell containing fibrin or collagen gel) is placed on a corner, spontaneous patterning is achieved within 1 second. Optimal dimensionless parameters required for successful capillary loading have been determined. To demonstrate the utility of the platform for 3D co-culture, angiogenesis experiments were performed by patterning HUVEC (human umbilical endothelial cells) and LF (lung fibroblasts) embedded in 3D fibrin gels. The angiogenic sprouts (with open lumen tip cells expressing junctional proteins) are comparable to those observed in PDMS based devices. The IMPACT device has the potential to provide a robust high-throughput experimental platform for vascularized microphysiological systems.

Microstructure guided multi-scale liquid patterning on an open surface.

Liquid patterning is a quintessential aspect in cell-based screening. While there are a variety of methods to handle microliquids utilizing surface treatments, complex microfluidic systems, and automated dispensing, most of the stated methods are both expensive and difficult to implement. Here, we present a fast multi-scale microliquid-patterning method on an open surface using embossed microstructures without surface modification. Arrays of micropillars can trap microliquids when a bulk drop is swept by an elastic sweeper on polystyrene (PS) substrates. The patterning mechanism on a basic form of a 2 × 2 rectangular array of circular pillars is analyzed theoretically and verified with experiments. Nanoliter-to-microliter volumes of liquids are patterned into various shapes by arranging the pillars based on the analysis. Furthermore, an array of geometrically modified pillars can capture approximately 8000 droplets on a large substrate (55 mm × 55 mm) in one step. Given the simplistic method of wipe patterning, the proposed platform can be utilized in both manual benchtop and automated settings. We will provide proof of concept experiments of single colony isolation using nanoliter-scale liquid patterning and of human angiogenic vessel formation using sequential patterning of microliter-scale liquids.