The investigation of human disease mechanisms is difficult due to the heterogeneity in gene expression and the physiological state of cells in a given population. In comparison to bulk cell measurements, single-cell measurement technologies can provide a better understanding of the interactions among molecules, organelles, cells, and the microenvironment, which can aid in the development of therapeutics and diagnostic tools. In recent years, single-cell technologies have become increasingly robust and accessible, although limitations exist. In this review, we describe the recent advances in single-cell technologies and their applications in single-cell manipulation, diagnosis, and therapeutics development.

Current Trends of Microfluidic Single-Cell Technologies.

To assemble microobjects including biological cells quickly and precisely, a fully automated pick-and-place operation is applied. In micromanipulation in liquid, the challenges include strong adhesion forces and high dynamic viscosity. To solve these problems, a reliable manipulation system and special releasing techniques are indispensable. A microhand having dexterous motion is utilized to grasp an object stably, and an automated stage transports the object quickly. To detach the object adhered to one of the end effectors, two releasing methods-local stream and a dynamic releasing-are utilized. A system using vision-based techniques for the recognition of two fingertips and an object, as well automated releasing methods, can increase the manipulation speed to faster than 800 ms/sphere with a 100% success rate (N = 100). To extend this manipulation technique, 2D and 3D assembly that manipulates several objects is attained by compensating the positional error. Finally, we succeed in assembling 80-120 µm of microbeads and spheroids integrated by NIH3T3 cells.

High-Speed Manipulation of Microobjects Using an Automated Two-Fingered Microhand for 3D Microassembly

We present a novel concept for the controlled trapping and releasing of beads and cells in a PDMS microfluidic channel without obstacles present around the particle or in the channel. The trapping principle relies on a two-level microfluidic configuration: a top main PDMS channel interconnected to a buried glass microchannel using round vias. As the fluidic resistances rule the way the liquid flows inside the channels, particles located in the streamlines passing inside the buried level are immobilized by the round via with a smaller diameter, leaving the object motionless in the upper PDMS channel. The particle is maintained by the difference of pressure established across its interface and acts as an infinite fluidic resistance, virtually cancelling the subsequent buried fluidic path. The pressure is controlled at the outlet of the buried path and three modes of operation of a trap are defined: idle, trapping and releasing. The pressure conditions for each mode are defined based on the hydraulic–electrical circuit equivalence. The trapping of polystyrene beads in a compact array of 522 parallel traps controlled by a single pressure was demonstrated with a trapping efficiency of 94%. Pressure conditions necessary to safely trap cells in holes of different diameters were determined and demonstrated in an array of 25 traps, establishing the design and operation rules for the use of planar hydrodynamic traps for biological assays.

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Planar hydrodynamic traps and buried channels for bead and cell trapping and releasing.

Patterning cells on a substrate is useful for cell assay and characterization. In this paper, a microchip employing negative dielectrophoresis was designed for efficient cell pattering. This chip consists of a 4-by-4 dot electrode array and the cell patterns are formed on a substrate lying on the microchip. To facilitate the microchip design, electric fields generated from the microchip were first simulated using software, and experiments were conducted to validate the performance of the microchip. Yeast cells suspending in the 6-aminohexanoic acid (AHA) solution were used in this study, and the effects of the cell medium, the voltage input and the voltage frequency were analyzed. The results confirm that cell patterns are successfully created with the microchip and this microchip offers an easy method to fabricate cell patterns on a substrate for characterization.

Characterization of a Microchip Device for Cell Patterning via Negative Dielectrophoresis

Single-cell analysis is becoming an indispensable tool in modern biological and medical research. Single-cell isolation is the key step for single-cell analysis. Single-cell printing shows several distinct advantages among the single-cell isolation techniques, such as precise deposition, high encapsulation efficiency, and easy recovery. Therefore, recent developments in single-cell printing have attracted extensive attention. We review herein the recently developed bioprinting strategies with single-cell resolution, with a special focus on inkjet-like single-cell printing. First, we discuss the common cell printing strategies and introduce several typical and advanced printing strategies. Then, we introduce several typical applications based on single-cell printing, from single-cell array screening and mass spectrometry-based single-cell analysis to three-dimensional tissue formation. In the last part, we discuss the pros and cons of the single-cell strategies and provide a brief outlook for single-cell printing.

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Advances in Single-Cell Printing

Parallel manipulation of single cells is important for reconstructing in vivo cellular microenvironments and studying cell functions. To manipulate single cells and reconstruct their environments, development of a versatile manipulation tool is necessary. In this study, we developed an array of hollow probes using microelectromechanical systems fabrication technology and demonstrated the manipulation of single cells. We conducted a cell aspiration experiment with a glass pipette and modeled a cell using a standard linear solid model, which provided information for designing hollow stepped probes for minimally invasive single-cell manipulation. We etched a silicon wafer on both sides and formed through holes with stepped structures. The inner diameters of the holes were reduced by SiO2 deposition of plasma-enhanced chemical vapor deposition to trap cells on the tips. This fabrication process makes it possible to control the wall thickness, inner diameter, and outer diameter of the probes. With the fabricated probes, single cells were manipulated and placed in microwells at a single-cell level in a parallel manner. We studied the capture, release, and survival rates of cells at different suction and release pressures and found that the cell trapping rate was directly proportional to the suction pressure, whereas the release rate and viability decreased with increasing the suction pressure. The proposed manipulation system makes it possible to place cells in a well array and observe the adherence, spreading, culture, and death of the cells. This system has potential as a tool for massively parallel manipulation and for three-dimensional hetero cellular assays.

Development and characterization of hollow microprobe array as a potential tool for versatile and massively parallel manipulation of single cells

High throughput reconstruction of in vivo cellular environments allows for efficient investigation of cellular functions. If one-side-open multi-channel microdevices are integrated with micropumps, the devices will achieve higher throughput in the manipulation of single cells while maintaining flexibility and open accessibility. This paper reports on the integration of a polydimethylsiloxane (PDMS) micronozzle array and bidirectional electrokinetic pumps driven by DC-biased AC voltages. Pt/Ti and indium tin oxide (ITO) electrodes were used to study the effect of DC bias and peak-to-peak voltage and electrodes in a low conductivity isotonic solution. The flow was bidirectionally controlled by changing the DC bias. A pump integrated with a micronozzle array was used to transport single HeLa cells into nozzle holes. The application of DC-biased AC voltage (100 kHz, 10 Vpp, and VDC: -4 V) provided a sufficient electroosmotic flow outside the nozzle array. This integration method of nozzle and pumps is anticipated to be a standard integration method. The operating conditions of DC-biased AC electrokinetic pumps in a biological buffer was clarified and found useful for cell manipulation.

https://www.mdpi.com/2072-666X/11/4/442/pdf

Scalable Parallel Manipulation of Single Cells Using Micronozzle Array Integrated with Bidirectional Electrokinetic Pumps.

A robust pick and placement operation of a single cell is necessary for efficient sample collection. Detection and manipulation of single cells requires minimum invasiveness. We report a less-invasive method for picking up and placing single cells using optical and electrical observations for robust cell manipulation. We measured the ionic current through a glass pipette during a cell capture and release operation to detect its capture. Trapping a cell on the pipette tip by suction decreased the current and allowed the detection of cell capture within 1 s. A time-series ionic current was sensitive to the location of a cell and effective at detecting a single cell. A time-series ionic current had a higher signal-to-noise ratio than time-series microscope images. Cell membrane integrity was analyzed at the different capturing and voltage conditions. Serum protein coating shows improvement of a cell release from a pipette tip. Measurement of trajectory and distance of a cell reveals that the movement depends on an ejection flow and the flow in a dish. We achieved a pick-up and placement operation for single cells that was compatible with an open-top microwell while performing observations using optical microscopy and measurements using an electrical current.

Pick-and-Place Operation of Single Cell Using Optical and Electrical Measurements for Robust Manipulation

Placement of different types of a single cell is necessary to measure life activity of living organism as close to original as possible. We propose an electrical detection and manipulation method of a single cell for parallel manipulation. To detect a cell capture, we measured a current through a glass pipette during cell capture and release. Trapping a cell on the pipette tip by suction at −5 kPa decreased the current by 1 nA in 0.4 s, which shows the cell trap can be detected in 1 s. Cell viability assay revealed that nine cells applied at 100 mV over 100 s were all alive. BSA coating improved a cell release from a pipette tip. Measurement of trajectory and distance of a cell revealed that the movement depended on an ejection flow and a flow in a dish.

Keita Kato

Papers

Development and characterization of hollow microprobe array as a potential tool for versatile and massively parallel manipulation of single cells

Scalable Parallel Manipulation of Single Cells Using Micronozzle Array Integrated with Bidirectional Electrokinetic Pumps.

Pick-and-Place Operation of Single Cell Using Optical and Electrical Measurements for Robust Manipulation

Electrical detection of single-cell trapping for manipulation in an array-based format