We introduce a microfluidic system for simultaneously measuring single cell mass and cell cycle progression over multiple generations. We use this system to obtain over 1,000 hours of growth data from mouse lymphoblast and pro-B-cell lymphoid cell lines. Cell lineage analysis revealed a decrease in the growth rate variability at the G1/S phase transition, which suggests the presence of a growth rate threshold for maintaining size homeostasis.

Direct observation of mammalian cell growth and size regulation

Exploring innovative solutions to improve the healthcare of the aging and diseased population continues to be a global challenge. Among a number of strategies toward this goal, tissue engineering and regenerative medicine (TERM) has gradually evolved into a promising approach to meet future needs of patients. TERM has recently received increasing attention in Asia, as evidenced by the markedly increased number of researchers, publications, clinical trials, and translational products. This review aims to give a brief overview of TERM development in Asia over the last decade by highlighting some of the important advances in this field and featuring major achievements of representative research groups. The development of novel biomaterials and enabling technologies, identification of new cell sources, and applications of TERM in various tissues are briefly introduced. Finally, the achievement of TERM in Asia, including important publications, representative discoveries, clinical trials, and examples of commercial products will be introduced. Discussion on current limitations and future directions in this hot topic will also be provided.

Tissue Engineering and Regenerative Medicine: Achievements, Future, and Sustainability in Asia.

Sub-micrometer particles (0.10-1.0 μm) are of great significance to study, e.g., microvesicles and protein aggregates are targets for therapeutic intervention, and sub-micrometer fluorescent polystyrene (PS) particles are used as probes for diagnostic imaging. Focusing of sub-micrometer particles - precisely control over the position of sub-micrometer particles in a tightly focused stream - has a wide range of applications in the field of biology, chemistry and environment, by acting as a prerequisite step for downstream detection, manipulation and quantification. Microfluidic devices have been attracting great attention as desirable tools for sub-micrometer particle focusing, due to their small size, low reagent consumption, fast analysis and low cost. Recent advancements in fundamental knowledge and fabrication technologies have enabled microfluidic focusing of particles at sub-micrometer scale in a continuous, label-free and high-throughput manner. Microfluidic methods for the focusing of sub-micrometer particles can be classified into two main groups depending on whether an external field is applied: 1) passive methods, which utilize intrinsic fluidic properties without the need of external actuation, such as inertial, deterministic lateral displacement (DLD), viscoelastic and hydrophoretic focusing; and 2) active methods, where external fields are used, such as dielectrophoretic, thermophoretic, acoustophoretic and optical focusing. This article mainly reviews the studies on the focusing of sub-micrometer particles in microfluidic devices over the past 10 years. It aims to bridge the gap between the focusing of micrometer and nanometer scale (1.0-100 nm) particles and to improve the understanding of development progress, current advances and future prospects in microfluidic focusing techniques.

Focusing of sub-micrometer particles in microfluidic devices.

Microfluidic applications such as active particle sorting or selective enrichment require particle classification techniques that are capable of working in real time. In this paper, we explore the use of neural networks for fast label-free particle characterization during microfluidic impedance cytometry. A recurrent neural network is designed to process data from a novel impedance chip layout for enabling real-time multiparametric analysis of the measured impedance data streams. As demonstrated with both synthetic and experimental datasets, the trained network is able to characterize with good accuracy size, velocity, and cross-sectional position of beads, red blood cells, and yeasts, with a unitary prediction time of 0.4 ms. The proposed approach can be extended to other device designs and cell types for electrical parameter extraction. This combination of microfluidic impedance cytometry and machine learning can serve as a stepping stone to real-time single-cell analysis and sorting.

A neural network approach for real-time particle/cell characterization in microfluidic impedance cytometry

Nano-drug delivery systems (NDDS) are functional drug-loaded nanocarriers extensively applied in the healthcare and pharmaceutical areas. Recently, microfluidics has been demonstrated as one of the most promising techniques to fabricate high-performance NDDS with uniform morphology, size and size distribution, reduced batch-to-batch variations and controllable drug delivering capacity. Here, a brief review of the microfluidic-mediated NDDS is presented. The fundamentals of microfluidics are first interpreted with an emphasis on the fluid characteristics, design and materials for microfluidic devices. Then a comprehensive and in-depth depiction of the microfluidic-mediated fabrications of controllable NDDS with well-tailored internal structures and integrated functions for controlled encapsulation and drug release are categorized and reviewed, with particular descriptions about the underlying formation mechanisms. Afterwards, recently appreciated representative applications of the microfluidic-mediated NDDS for delivering multiple drugs are systematically summarized. Finally, conclusions and perspectives on further advancing the microfluidic-mediated NDDS toward more powerful and versatile platforms for therapeutic applications are discussed.

Microfluidic-mediated nano-drug delivery systems: from fundamentals to fabrication for advanced therapeutic applications

Effective and high-throughput cell sorting are critical technologies in single cell analysis in biology, medicine and clinical applications. Especially for single cell analysis requiring high accuracy, multiple sorting mechanisms are required, and they need to be well organized into a system based on the sorting ability. Therefore, we classify most recent methods of microfluidic cell sorting as precise or large amount sorting methods based on the ability for sorting the number of cells. Precise sorting applies an instantaneous force (e.g. acoustic, electric, optical, and jet force) to displace cells a short distance to isolate them one by one with relatively high accuracy. Large amount methods use a continuous force (e.g. acoustic, electric, magnetic and passive force) to arrange, separate and purify cell populations with relatively high-throughput. By combining precise sorting and large amount sorting methods, it is possible to achieve a high-accuracy sorting system with high-throughput. Therefore, in this review, we summarized merits and demerits of these advanced technologies of sorting (focusing on those devices in the last five years that have features of high-throughput, portability and high efficiency) to suggest some suitable combinations of large amount and precise sorting methods; then we discussed the direction of single cell sorting and the prospects of the combined microchips.

Recent advances in microfluidic cell sorting systems

Glass is a common material used in various microsystems including sensors, actuators, and microfluidic devices due to its extraordinary properties. However, lack of flexibility and elasticity limits its potential for further applications. To solve this issue, thinning the thick glass plate to a few microns is a solution. However, conventional methods for thinning thick glass plates are complex, risky, time-consuming, and difficult for implementation at the laboratory level. This paper reports an efficient and convenient fabrication method for an independent ultra-thin glass sheet (UTGS) (≤ 3 μm, the world’s thinnest) by utilizing a weight-controlled load-assisted (WCL-assisted) thermal stretching. Unlike other methods requiring a full melting of the glass material, the key point of the proposed method is using a WCL to precisely stretch a commercial thick glass plate to achieve the desired glass sheet thickness. To fabricate UTGSs of the desired thicknesses, experimental conditions including temperatures (630°C–700°C), cooling times (1.5 h–2.5 h), and weights of loads (1 g–9 g) were investigated. Furthermore, the potential advantage of using the fabricated UTGS for indicator applications in micro-systems was proven by fabricating a pressure indicator having higher sensitivity than presently available devices. For the same working conditions, the maximum sensitivity of the new indicator was improved by 86.8 % compared with the sensitivity previously obtained.

Fabrication of ultra-thin glass sheet by weight-controlled load-assisted precise thermal stretching

Microscopic impedance cytometry for quantifying single cell shape

We demonstrated a self-driven chemical micropump powered by self-oscillating motions of synthetic polymer gel converting chemical energy into displacements of a thin polydimethylsiloxane (PDMS) sheet to produce a directional flow via micro check valves. The gel oscillations induced by the oscillatory Belousov-Zhabotinsky (BZ) reaction generate large volume change. To effectively exploit this volume change, gel bars were used as drivers. They were sandwiched by a fixed cover glass and a push-bar to communicate the expanding (pushing) force of the gel bars to the pumping chamber. At 25 °C, this gel micropump generated a 0.07 mN stroke force and a 0.28 μL/min positive net flow in the microchannel. Additionally, the flow rate of this gel micropump was increased as the temperature was raised from 20 to 30 °C. Compared to existing chemical micropumps, this gel-based micropump provides advantages of self-oscillating property, easy refueling and thermal controllability. This gel-based soft micropump would be used as wearable or medical devices exploiting the flexibility and quietness.

A chemical micropump actuated by self-oscillating polymer gel

Bio-actuators that use insect muscular tissue have attracted attention from researchers worldwide because of their small size, self-motive property, self-repairer ability, robustness, and the need for less environment management than mammalian cells. To demonstrate the potential of insect muscular tissue for use as bio-actuators, three types of these robots, a pillar actuator, a walker, and a twizzer, have been designed and fabricated. However, a model of an insect muscular tissue-powered swimming robot that is able to float and swim in a solution has not yet been reported. Therefore, in this paper, we present a prototype of an insect muscular tissue-powered autonomous micro swimming robot that operates at room temperature and requires no temperature and pH maintenance. To design a practical robot body that is capable of swimming by using the force of the insect dorsal vessel (DV), we first measured the contraction force of the DV. Then, the body of the swimming robot was designed, and the design was confirmed by a simulation that used the condition of measured contraction force. After that, we fabricated the robot body using polydimethylpolysiloxane (PDMS). The PDMS body was obtained from a mold that was fabricated by a stereo lithography method. Finally, we carefully attached the DV to the PDMS body to complete the assembly of the swimming robot. As a result, we confirmed the micro swimming robot swam autonomously at an average velocity of 11.7 μm/s using spontaneous contractions of the complete insect DV tissue. These results demonstrated that the insect DV has potential for use as a bio-actuator for floating and swimming in solution.

Yigang Shen

Papers

Recent advances in microfluidic cell sorting systems

Fabrication of ultra-thin glass sheet by weight-controlled load-assisted precise thermal stretching

Microscopic impedance cytometry for quantifying single cell shape

A chemical micropump actuated by self-oscillating polymer gel

Insect Muscular Tissue-Powered Swimming Robot