Keekyoung Kim

Nanonewton force-controlled manipulation of biological cells using a monolithic MEMS microgripper with two-axis force feedback

As mechanical end-effectors, microgrippers enable the pick–transport–place of micrometer-sized objects, such as manipulation and positioning of biological cells in an aqueous environment. This paper reports on a monolithic MEMS-based microgripper with integrated force feedback along two axes and presents the first demonstration of force- controlled micro-grasping at the nanonewton force level. The system manipulates highly deformable biomaterials (porcine interstitial cells) in an aqueous environment using a microgripper that integrates a V-beam electrothermal microactuator and two capacitive force sensors, one for contact detection (force resolution: 38.5 nN) and the other for gripping force measurements (force resolution: 19.9 nN). The MEMS-based microgripper and the force control system experimentally demonstrate the capability of rapid contact detection and reliable force-controlled micrograsping to accommodate variations in size and mechanical properties of objects with a high reproducibility.

3D bioprinting for engineering complex tissues.

Bioprinting is a 3D fabrication technology used to precisely dispense cell-laden biomaterials for the construction of complex 3D functional living tissues or artificial organs. While still in its early stages, bioprinting strategies have demonstrated their potential use in regenerative medicine to generate a variety of transplantable tissues, including skin, cartilage, and bone. However, current bioprinting approaches still have technical challenges in terms of high-resolution cell deposition, controlled cell distributions, vascularization, and innervation within complex 3D tissues. While no one-size-fits-all approach to bioprinting has emerged, it remains an on-demand, versatile fabrication technique that may address the growing organ shortage as well as provide a high-throughput method for cell patterning at the micrometer scale for broad biomedical engineering applications. In this review, we introduce the basic principles, materials, integration strategies and applications of bioprinting. We also discuss the recent developments, current challenges and future prospects of 3D bioprinting for engineering complex tissues. Combined with recent advances in human pluripotent stem cell technologies, 3D-bioprinted tissue models could serve as an enabling platform for high-throughput predictive drug screening and more effective regenerative therapies.

Directed endothelial cell morphogenesis in micropatterned gelatin methacrylate hydrogels

Engineering of organized vasculature is a crucial step in the development of functional and clinically relevant tissue constructs. A number of previous techniques have been proposed to spatially regulate the distribution of angiogenic biomolecules and vascular cells within biomaterial matrices to promote vascularization. Most of these approaches have been limited to two-dimensional (2D) micropatterned features or have resulted in formation of random vasculature within three-dimensional (3D) microenvironments. In this study, we investigate 3D endothelial cord formation within micropatterned gelatin methacrylate (GelMA) hydrogels with varying geometrical features (50-150 μm height). We demonstrated the significant dependence of endothelial cells proliferation, alignment and cord formation on geometrical dimensions of the patterned features. The cells were able to align and organize within the micropatterned constructs and assemble to form cord structures with organized actin fibers and circular/elliptical cross-sections. The inner layer of the cord structure was filled with gel showing that the micropatterned hydrogel constructs guided the assembly of endothelial cells into cord structures. Notably, the endothelial cords were retained within the hydrogel microconstructs for all geometries after two weeks of culture; however, only the 100 μm-high constructs provided the optimal microenvironment for the formation of circular and stable cord structures. Our findings suggest that endothelial cord formation is a preceding step to tubulogenesis and the proposed system can be used to develop organized vasculature for engineered tissue constructs.

Nanonewton Force Sensing and Control in Microrobotic Cell Manipulation

Cellular force sensing and control techniques are capable of enhancing the dexterity and reliability of microrobotic cell manipulation systems. In this paper we present two experimental techniques for nanonewton force sensing and control in microrobotic cell manipulation. A vision-based cellular force sensing approach, including a microfabricated elastic cell holding device and a sub-pixel visual tracking algorithm, was developed for resolving forces down to 3.7 nN during microrobotic mouse embryo injection. The technique also experimentally demonstrated that the measured mechanical difference could be useful for in situ differentiation of healthy mouse embryos from those with compromised developmental competence without requiring a separate mechanical characterization process. Centered upon force-controlled microrobotic cell manipulation, this paper also presents nanonewton force-controlled micrograsping of interstitial cells using a microelectromechanical systems (MEMS)-based microgripper with integrated two-axis force feedback. On-chip force sensors are used for detecting contact between the microgripper and cells to be manipulated (resolution: 38.5 nN at 15Hz) and sensing gripping forces (resolution: 19.9 nN at 15Hz) during force-controlled grasping. The experimental results demonstrate that the microgripper and the control system are capable of rapid contact detection and reliable force-controlled micrograsping to accommodate variations in size and stiffness of cells with a high degree of reproducibility.

A MEMS stage for 3-axis nanopositioning

Applications in micro- and nanotechnologies require millimeter-sized devices that are capable of 3-axis positioning with motion ranges of micrometers and resolutions of nanometers. This paper reports on the design, fabrication and testing of a MEMS-based 3-axis positioning stage. In-plane (comb-drive) and out-of-plane (parallel-plate) electrostatic actuators are employed for driving the stage to move independently along the XYZ directions, by ±12.5 µm in the X and Y directions at an actuation voltage of 30 V and by 3.5 µm in the Z direction at 14.8 V. The structures are designed to achieve highly decoupled motions by effectively suppressing cross-axis motion coupling. The open-loop positioning repeatability is determined to be better than 17.3 nm along all three axes.

Adipose-Derived Stem Cells for Tissue Engineering and Regenerative Medicine Applications

Adipose-derived stem cells (ASCs) are a mesenchymal stem cell source with properties of self-renewal and multipotential differentiation. Compared to bone marrow-derived stem cells (BMSCs), ASCs can be derived from more sources and are harvested more easily. Three-dimensional (3D) tissue engineering scaffolds are better able to mimic the in vivo cellular microenvironment, which benefits the localization, attachment, proliferation, and differentiation of ASCs. Therefore, tissue-engineered ASCs are recognized as an attractive substitute for tissue and organ transplantation. In this paper, we review the characteristics of ASCs, as well as the biomaterials and tissue engineering methods used to proliferate and differentiate ASCs in a 3D environment. Clinical applications of tissue-engineered ASCs are also discussed to reveal the potential and feasibility of using tissue-engineered ASCs in regenerative medicine.

Investigation of mechanical properties of soft hydrogel microcapsules in relation to protein delivery using a MEMS force sensor.

This article reports the investigation of mechanical properties of alginate-chitosan microcapsules and the relation to protein delivery. For microscale compression testing, a system based on a microelectromechanical systems (MEMS) capacitive force sensor was developed. The bulk microfabricated capacitive force sensors are capable of resolving forces up to 110 microN with a resolution of 33.2 nN along two independent axes. The monolithic force sensors were directly applied to characterizing mechanical properties of soft hydrogel microparticles without assembling additional end-effectors. Protein-loaded alginate-chitosan microcapsules of approximately 20 mum in diameter were prepared by an emulsion-internal gelation-polyelectrolyte coating method. Youngs modulus values of individual microcapsules with 1, 2, and 3% chitosan coating were determined through microscale compression testing in both distilled deionized (DDI) water and pH 7.4 phosphate-buffered saline (PBS). Protein release rates were also determined in DDI water and PBS. Finally, protein release rates were correlated with mechanical properties of the microcapsules.

Microfluidics-Assisted Fabrication of Gelatin-Silica Core–Shell Microgels for Injectable Tissue Constructs

Microfabrication technology provides a highly versatile platform for engineering hydrogels used in biomedical applications with high-resolution control and injectability. Herein, we present a strategy of microfluidics-assisted fabrication photo-cross-linkable gelatin microgels, coupled with providing protective silica hydrogel layer on the microgel surface to ultimately generate gelatin-silica core–shell microgels for applications as in vitro cell culture platform and injectable tissue constructs. A microfluidic device having flow-focusing channel geometry was utilized to generate droplets containing methacrylated gelatin (GelMA), followed by a photo-cross-linking step to synthesize GelMA microgels. The size of the microgels could easily be controlled by varying the ratio of flow rates of aqueous and oil phases. Then, the GelMA microgels were used as in vitro cell culture platform to grow cardiac side population cells on the microgel surface. The cells readily adhered on the microgel surface and proliferated over time while maintaining high viability (∼90%). The cells on the microgels were also able to migrate to their surrounding area. In addition, the microgels eventually degraded over time. These results demonstrate that cell-seeded GelMA microgels have a great potential as injectable tissue constructs. Furthermore, we demonstrated that coating the cells on GelMA microgels with biocompatible and biodegradable silica hydrogels via sol–gel method provided significant protection against oxidative stress which is often encountered during and after injection into host tissues, and detrimental to the cells. Overall, the microfluidic approach to generate cell-adhesive microgel core, coupled with silica hydrogels as a protective shell, will be highly useful as a cell culture platform to generate a wide range of injectable tissue constructs.

Micronewton force-controlled manipulation of biomaterials using a monolithic MEMS microgripper with two-axis force feedback

This paper presents the first demonstration of force-controlled micrograsping at the microNewton force level. The system manipulates highly deformable biomaterials (hydrogel microcapsules and biological cells) in an aqueous environment using a MEMS-based microgripper with integrated force feedback along two axes. The microgripper integrates an electrothermal V-beam microactuator and two capacitive force sensors, one for contact detection (force resolution: 38.5 nN) and the other for gripping force measurements (force resolution: 19.9 nN). The MEMS-based microgripper and the force control system experimentally demonstrate the capability of rapid contact detection and reliable force-controlled micrograsping to accommodate variations in sizes and mechanical properties of objects with a high reproducibility. Cell viability testing validated that the temperature at gripping arm tips does not exceed 50degC.

Calibration of Multi-Axis MEMS Force Sensors Using the Shape-From-Motion Method

Precise calibration of multi-axis microelectromechanical systems (MEMS) force sensors is difficult for several reasons, including the need to apply many known force vectors at precise orientations at the micro- and nanoNewton (nN) force scales, and the risk of damaging the small, fragile microdevices. To tackle these challenges, this paper introduces the shape-from-motion calibration method. A new design of a two-axis MEMS capacitive force sensor with high linearity and nN resolutions is presented. Structural-electrostatic coupled-field simulations are conducted in order to optimize the sensor design. The designed sensor is calibrated with the shape-from-motion method, the least-squares method as well as the gravity-based method for comparison purposes. Calibration results demonstrate that the shape-from-motion method provides a rapid, practical, and accurate technique for calibrating multi-axis MEMS sensors