Shuxun Chen

Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies.

Sorting (or isolation) and manipulation of rare cells with high recovery rate and purity are of critical importance to a wide range of physiological applications. In the current paper, we report on a generic single cell manipulation tool that integrates optical tweezers and microfluidic chip technologies for handling small cell population sorting with high accuracy. The laminar flow nature of microfluidics enables the targeted cells to be focused on a desired area for cell isolation. To recognize the target cells, we develop an image processing methodology with a recognition capability of multiple features, e.g., cell size and fluorescence label. The target cells can be moved precisely by optical tweezers to the desired destination in a noninvasive manner. The unique advantages of this sorter are its high recovery rate and purity in small cell population sorting. The design is based on dynamic fluid and dynamic light pattern, in which single as well as multiple laser traps are employed for cell transportation, and a recognition capability of multiple cell features. Experiments of sorting yeast cells and human embryonic stem cells are performed to demonstrate the effectiveness of the proposed cell sorting approach.

Probing the mechanobiological properties of human embryonic stem cells in cardiac differentiation by optical tweezers

Human embryonic stem cells (hESC) and hESC-derived cardiomyocytes (hESC-CM) hold great promise for the treatment of cardiovascular diseases. However the mechanobiological properties of hESC and hESC-CM remains elusive. In this paper, we examined the dynamic and static micromechanical properties of hESC and hESC-CM, by manipulating via optical tweezers at the single-cell level. Theoretical approaches were developed to model the dynamic and static mechanical responses of cells during optical stretching. Our experiments showed that the mechanical stiffness of differentiated hESC-CM increased after cardiac differentiation. Such stiffening could associate with increasingly organized myofibrillar assembly that underlines the functional characteristics of hESC-CM. In summary, our findings lay the ground work for using hESC-CMs as models to study mechanical and contractile defects in heart diseases.

Laser-induced fusion of human embryonic stem cells with optical tweezers

We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

Cell manipulation tool with combined microwell array and optical tweezers for cell isolation and deposition

Isolation from rare cells and deposition of sorted cells with high accuracy for further study are critical to a wide range of biomedical applications. In the current paper, we report an automated cell manipulation tool with combined optical tweezers and a uniquely designed microwell array, which functions for recognition, isolation, assembly, transportation and deposition of the interesting cells. The microwell array allows the passive hydrodynamic docking of cells, while offering the opportunity to inspect the interesting cell phenotypes with high spatio-temporal resolution based on the flexible image processing technique. In addition, dynamic and parallel cell manipulation in three dimensions can realize the target cell levitation from microwell and pattern assembly with multiple optical traps. Integrated with the programmed motorized stage, the optically levitated and assembled cells can be transported and deposited to the predefined microenvironment, so the tool can facilitate the integration of other on-chip functionalities for further study without removing these isolated cells from the chip. Experiments on human embryonic stem cells and yeast cells are performed to demonstrate the effectiveness of the proposed cell manipulation tool. Besides the application to cell isolation and deposition, three other biological applications with this tool are also presented.

A High-Throughput Automated Microinjection System for Human Cells With Small Size

This paper presents the development of an automated microinjection system with high productivity for small cells. Compared with many existing microinjection systems that are primarily designed for relatively large-scaled biological samples, the reported technology will enable an automated injection into the cells with a diameter smaller than 25 μm, which is the typical size of many human cells. Microfluidic technology has been used to design a vacuum-based cell-holding device for immobilizing cells. The cell holder employs hundreds of regular-shaped channels, and each channel is occupied by one cell. The vision-based position-tracking method is used to recognize and position the target cells automatically, in which the cell position is measured by edge template-matching method, and a bright field image is used to simplify the recognition process. A 3-DOF microrobotic system mounted with a micropipette is used to conduct cell injection task with the speed of 35 cells/min and the precision of 0.2 μm. Injection experiments on human foreskin fibroblast and human embryonic stem cell-derived ventricular cardiomyocyte were performed to demonstrate the effectiveness of the developed system. More than 1000 cells were injected with fluorescent markers. Experimental results showed that the developed injection system exhibits high productivity and accuracy, and results in a good cell survival rate after injection.

Automated Transportation of Multiple Cell Types Using a Robot-Aided Cell Manipulation System With Holographic Optical Tweezers

Transferring multiple cell types with high precision and efficiency has become increasingly important for developing cell-based assays. In this study, an enabling technology is proposed for simultaneous automated transportation of multiple cell types utilizing a robot-aided cell manipulation system equipped with holographic optical tweezers. The dynamics of a trapped cell is initially analyzed. A control constraint is introduced to confine the offset of cells within the optical trap to prevent cells from escaping the trap during transportation. Unlike existing methods determining the critical offset through manual calibration for only a particular cell type, this proposed approach can automatically derive and apply the control constraint to multiple cell types with different radii. A controller is then developed for automated transportation of multiple cell types with different sizes in which exact values of model parameters, such as trapping stiffness and drag coefficient, are not required. Experiments are finally performed on the transportation of yeast cells and osteoblast-like MC3T3-E1 cells to demonstrate the effectiveness of the proposed approach.

In Vivo Manipulation of Single Biological Cells With an Optical Tweezers-Based Manipulator and a Disturbance Compensation Controller

In vivo manipulation of biological cells has attracted considerable attention in recent years. This process is particularly useful for precision medicine, such as cancer target therapy. Robotics technology is becoming necessary to stably and effectively manipulate and control single target cells in a complex in vivo environment. This paper presents a robot-aided optical tweezers-based manipulation technology that serves a function in the transport of single biological cells in vivo. An enhanced disturbance compensation controller is developed to minimize the effect of fluids (e.g., blood flow) on the cell. The method has exhibited advantages of flexibility in adjusting cell tracking trajectory online and the capability to minimize steady-state error and eliminate overshoot. Simulations and experiments of tracking single target cells in living zebrafish embryos have demonstrated the effectiveness of the proposed approach in a dynamic in vivo environment.

Microfluidic single-cell array platform enabling week-scale clonal expansion under chemical/electrical stimuli

Single-cell culture represents the most straightforward method for investigating cellular heterogeneity. In this paper, we present a novel microfluidic platform that can individually array and culture hundreds of cells under chemical and electrical stimuli for week-scale characterization. Single cells can be deterministically and gently captured in a microchamber array on the proposed platform. The size of the microchamber can be adjusted to fit different cell culture times, and this characteristic enables remarkable scalability. Transparent indium tin oxide microelectrodes were integrated with the single-cell array platform for on-chip electrical stimuli. The platform exhibited nearly 90% single-cell efficiency and facilitated week-scale clonal expansion of different types of single cells. Chemical and electrical stimuli affected proliferation and differentiation of MC 3T3-E1 cells were examined on the chip prototype that contained 416 (32 rows × 13 columns) microchambers, and each microchamber had 1 mm ...

Fusion with stem cell makes the hepatocellular carcinoma cells similar to liver tumor-initiating cells

BackgroundCell fusion is a fast and highly efficient technique for cells to acquire new properties. The fusion of somatic cells with stem cells can reprogram somatic cells to a pluripotent state. Our research on the fusion of stem cells and cancer cells demonstrates that the fused cells can exhibit stemness and cancer cell-like characteristics. Thus, tumor-initiating cell-like cells are generated.MethodsWe employed laser-induced single-cell fusion technique to fuse the hepatocellular carcinoma cells and human embryonic stem cells (hESC). Real-time RT-PCR, flow cytometry and in vivo tumorigenicity assay were adopted to identify the gene expression difference.ResultsWe successfully produced a fused cell line that coalesces the gene expression information of hepatocellular carcinoma cells and stem cells. Experimental results showed that the fused cells expressed cancer and stemness markers as well as exhibited increased resistance to drug treatment and enhanced tumorigenesis.ConclusionsFusion with stem cells transforms liver cancer cells into tumor initiating-like cells. Results indicate that fusion between cancer cell and stem cell may generate tumor initiating-like cells.

Development of a high throughput robot-aided cell injection system for human cells

Few of the current injection technologies can be applied to those human cells whose diameters are ranged about 10-25 (im only. This paper reports our most recent effort in developing a robot-aided microinjection system to solve the challenging problem of automated injection on human cells. A unique microfluidic cell holding chip is designed and fabricated to trap the single cells in the predefined docking area. Imaging processing technique is used to recognize automatically the target cells to be injected. A microrobot system equipped with a micropipette is used to perform the injection tasks on these target cells. Injection experiments on human embryonic stem cells (hESCs) (ranged about 17-25μm) are performed to demonstrate the effectiveness of the proposed microinjection system.