Microfluidic technology has been extensively employed in biology and medicine since the field emerged in the 1990s. By utilizing microfluidic approaches, a variety of vascular system-related structures and functions have been mimicked on in vitro platforms. Herein, we begin by introducing microfluidic circulatory devices for the study of two-dimensional (2D) endothelial cells culture. Next, we focus on recent progress on on-chip mimicry of native vasculature, specifically generation of complex three-dimensional (3D) structures within cell-laden hydrogels using microfluidics and self-assembly-based methods. The utilization of microfluidic technology will facilitate the construction of progressively biomimetic in vitro models that have great potential in complementing existing animal models. We envision such platforms to be utilized in a wide range of applications involving vascular systems, including microphysiological studies, drug screening, and disease modeling.

The human circulatory system comprises a complex network of blood vessels interconnecting biologically relevant organs and a heart driving blood recirculation throughout this system. Recreating this system in vitro would act as a bridge between organ-on-a-chip and "body-on-a-chip" and advance the development of in vitro models. Here, we present a microfluidic circulatory system integrated with an on-chip pressure sensor to closely mimic human systemic circulation in vitro. A cardiac-like on-chip pumping system is incorporated in the device. It consists of four pumping units and passive check valves, which mimic the four heart chambers and heart valves, respectively. Each pumping unit is independently controlled with adjustable pressure and pump rate, enabling users to control the mimicked blood pressure and heartbeat rate within the device. A check valve is located downstream of each pumping unit to prevent backward leakage. Pulsatile and unidirectional flow can be generated to recirculate within the device by programming the four pumping units. We also report an on-chip capillary-assisted pressure sensor to monitor the pressure inside the device. One end of the capillary was placed in the measurement region, while the other end was sealed. Time-dependent pressure changes were measured by recording the movement of the liquid-gas interface in the capillary and calculating the pressure using the ideal gas law. The sensor covered the physiologically relevant blood pressure range found in humans (0-142.5 mmHg) and could respond to 0.2 s actuation time. With the aid of the sensor, the pressure inside the device could be adjusted to the desired range. As a proof of concept, human normal left ventricular and arterial pressure profiles were mimicked inside this device. Human umbilical vein endothelial cells (HUVECs) were cultured on chip and cells can respond to mechanical forces generated by arterial-like flow patterns.

Single-cell measurements have played a critical role in revealing the complex signaling dynamics and heterogeneity present in cells, but there is still much to learn. Measuring samples from bulk populations of cells often masks the information and dynamics present in subsets of cells. Common single-cell protein studies rely on fluorescent microscopy and flow cytometry but are limited in multiplexing ability owing to spectral overlap. Recently, technology advancements in single-cell proteomics have allowed highly multiplexed measurement of multiple parameters simultaneously by using barcoded microfluidic enzyme-linked immunosorbent assays and mass cytometry techniques. In this review, we will describe recent work around multiparameter single-cell protein measurements and critically analyze the techniques.

The diffusion of molecules such as nutrients and oxygen through densely packed cells is impeded by blockage and consumption by cells, resulting in a limited depth of penetration. This has been a major hurdle to a bulk (3-D) culture. Great efforts have been made to develop methods for generating branched microchannels inside hydrogels to support mass exchange inside a bulk culture. These previous attempts faced a common obstacle: researchers tried to fabricate microchannels with gels already loaded with cells, but the fabrication procedures are often harmful to the embedded cells. Herein, we present a universal strategy to create microchannels in different types of hydrogels, which effectively avoids cell damage. This strategy is based on a freestanding alginate 3-D microvascular network prepared by in-situ generation of copper ions from a sacrificial copper template. This alginate network could be used as implants to create microchannels inside different types of hydrogels. This approach effectively addresses the issue of cell damage during microfabrication and made it possible to create microchannels inside different types of gels. The microvascular network produced with this method is (1) strong enough to allow handling, (2) biocompatible to allow cell culturing, and (3) appropriately permeable to allow diffusion of small molecules, while sufficiently dense to prevent blocking of channels when embedded in different types of gels. In addition, composite microtubules could be prepared by simply pre-loading other materials, e.g., particles and large biomolecules, in the hydrogel. Compared with other potential strategies to fabricate freestanding gel channel networks, our method is more rapid, low-cost and scalable due to parallel processing using an industrially mass-producible template. We demonstrated the use of such vascular networks in creating microchannels in different hydrogels and composite gels, as well as with a cell culture in a nutrition gradient based on microfluidic diffusion. In this way, the freestanding hydrogel vascular network we produced is a universal functional unit that can be embedded in different types of hydrogel; users will be able to adopt this strategy to achieve vascular mass exchange in the bulk culture without changing their current protocol. The method is readily implementable to applications in vascular tissue regeneration, drug discovery, 3-D culture, etc.

The fabrication of microfluidic chips can be simplified and accelerated by three-dimensional (3D) printing. However, all of the current designs of 3D printed microchips require off-chip bulky equipment to operate, which hindered their applications in the point-of-care (POC) setting. In this work, we demonstrate a new class of movable 3D printed microfluidic chip components, including torque-actuated pump and valve, rotary valve, and pushing valve, which can be operated manually without any off-chip bulky equipment such as syringe pump and gas pressure source. By integrating these components, we developed a user-friendly 3D printed chip that can perform general colorimetric assays. Protein quantification was performed on artificial urine samples as a proof-of-concept model with a smartphone used as the imaging platform. The protein was quantified linearly and was within the physiologically relevant range for humans. We believe that the demonstrated components and designs can expand the functionalities and potential applications of 3D printed microfluidic chip and thus provoke more investigation on manufacturing lab-on-a-chip devices by 3D printers.

An ultra-fast, extremely cost-effective, and environmentally friendly method was developed for fabricating flexible microfluidic chips with plastic membranes. With this method, we could fabricate plastic microfluidic chips rapidly (within 12 seconds per piece) at an extremely low cost (less than $0.02 per piece). We used a heated perfluoropolymer perfluoroalkoxy (often called Teflon PFA) solid stamp to press a pile of two pieces of plastic membranes, low density polyethylene (LDPE) and polyethylene terephthalate (PET) coated with an ethylene-vinyl acetate copolymer (EVA). During the short period of contact with the heated PFA stamp, the pressed area of the membranes permanently bonded, while the LDPE membrane spontaneously rose up at the area not pressed, forming microchannels automatically. These two regions were clearly distinguishable even at the micrometer scale so we were able to fabricate microchannels with widths down to 50 microns. This method combines the two steps in the conventional strategy for microchannel fabrication, generating microchannels and sealing channels, into a single step. The production is a green process without using any solvent or generating any waste. Also, the chips showed good resistance against the absorption of Rhodamine 6G, oligonucleotides, and green fluorescent protein (GFP). We demonstrated some typical microfluidic manipulations with the flexible plastic membrane chips, including droplet formation, on-chip capillary electrophoresis, and peristaltic pumping for quantitative injection of samples and reagents. In addition, we demonstrated convenient on-chip detection of lead ions in water samples by a peristaltic-pumping design, as an example of the application of the plastic membrane chips in a resource-limited environment. Due to the high speed and low cost of the fabrication process, this single-step method will facilitate the mass production of microfluidic chips and commercialization of microfluidic technologies.

Hydrogels, with a complicated 3D structure, were fabricated by replicating the structure of a 3D printed master.

In this work, we developed a convenient, one-step soft-lithographic-based molding technique for molding truly 3D microfluidic channels in polydimethylsiloxane (PDMS) by overcoming two grand challenges. We optimized the post-treatment conditions for 3D-printed resin structures to facilitate the use of them as masters for PDMS replica molding. What is more important, we demonstrated a novel method for single-step molding from 3D-printed microstructures to generate truly 3D microfluidic networks easily. With this technique, we fabricated some key, functional 3D microfluidic structures and components including a basket-weaving network, a 3D chaotic advective mixer and microfluidic peristaltic valves. Furthermore, an interesting “injection-on-demand” microfluidic device was also demonstrated. Our technique offers a simple, fast route to the fabrication of 3D microfluidic chips in a short time without clean-room facilities.

Microgrooved thin PLGA film (≈30 μm) is successfully fabricated on a Teflon mold, which could be readily peeled off and is used for the construction of a biomimetic cardiac patch. The contraction of it is studied with optical mapping on transmembrane action potential. Our results suggest that steady-state contraction could be easily established on it under regular electrical stimuli. Besides, the biomimetic cardiac patch recapitulates the anisotropic electrophysiological feature of native cardiac tissue and is much more refractory to premature stimuli than the random one constructed with non-grooved PLGA film, as proved by the reduced incidence of arrhythmia. Considering the good biocompatibility of PLGA as demonstrated in our study and the biodegradability of it, our biomimetic cardiac patch may find applications in the treatment of myocardial infarction. Moreover, the Teflon mold could be applied in the fabrication of various scaffolds with fine features for other tissues.

Microfluidic devices have been extensively used in single-cell assays. However, most of them have complicated structures (multiple layers, valves, and channels) and require the assistance of a pump or pressure-controlling system. In this paper, we present a facile centrifugation-assisted single-cell trapping (CAScT) approach based on a truncated cone-shaped microwell array (TCMA) chip for real-time observation of cellular apoptosis. Our method requires neither a pump nor a pressure-controlling system, and it greatly reduces the complexity of other cell-trapping devices. This method is so fast and efficient that single-cell occupancy could reach approximately 90% within a few seconds. Combined with modern fluorescence microscopy, CAScT makes the highly ordered and addressable TCMA a high-throughput platform (10(4)-10(5) single-cell trapping sites per cm(2)) for single-cell analysis. Cells trapped in it could be exposed to various chemicals by directly immersing it in bulk solutions without the significant loss of cells due to the truncated cone shape of the microwells. As a proof of concept, we demonstrated the ability of our chip for the real-time observation of the apoptosis of single HeLa cells induced by the common anticancer drug doxorubicin. This simple, robust, and efficient approach possesses great potential in diverse applications, such as drug screening, biosensing, and fundamental biological research.