Mechanical Manipulation and Characterization of Biological Cells

Mechanical manipulation and characterization of an individual biological cell is currently one of the most exciting research areas in the field of medical robotics. Single cell manipulation is an important process in intracytoplasmic sperm injection (ICSI), pro-nuclei DNA injection, gene therapy, and other biomedical areas. However, conventional cell manipulation requires long training and the success rate depends on the experience of the operator. The goal of this research is to address the drawbacks of conventional cell manipulation by using force and vision feedback for cell manipulation tasks. We hypothesize that force feedback plays an important role in cell manipulation and possibly helps in cell characterization. This dissertation will summarize our research on: 1) the development of force and vision feedback interface for cell manipulation, 2) human subject studies to evaluate the addition of force feedback for cell injection tasks, 3) the development of haptics-enabled atomic force microscope system for cell indentation tasks, 4) appropriate analytical model for characterizing the mechanical property of mouse embryonic stem cells (mESC) and 5) several indentation studies on mESC to determine the mechanical property of undifferentiated and early differentiating (6 days under differentiation conditions) mESC. Our experimental results on zebrafish egg cells show that a system with force feedback capability when combined with vision feedback can lead to potentially higher success rates in cell injection tasks. Using this information, we performed experiments on mESC using the AFM to understand their characteristics in the undifferentiated pluripotent state as well as early differentiating state. These experiments were done on both live as well as fixed cells to understand the correlation between the two during cell indentation studies. Our results show that the mechanical property of undifferentiated mESC differs from early differentiating (6th day) mESC in both live and fixed cells. Thus, we hypothesize that mechanical characterization studies will potentially pave the way for developing a high throughput system with force feedback capability, to understand and predict the differentiation path a particular pluripotent cell will follow. This finding could also be used to develop improved methods of targeted cellular differentiation of stem cells for therapeutic and regenerative medicine

Evaluating the role of force feedback for biomanipulation tasks

Paper presented at the IEEE Virtual Reality, Haptics Symposium and Symposium on 3D User Interface, Alexandria, VA.Conventional cell manipulation techniques do not have the ability to provide force feedback to an operator. Poor control of cell injection force is one of the primary reasons for low success rates in cell injection and transgenesis in particular. Therefore, there exists a need to incorporate force feedback into a cell injection system. We have developed an automated cell injection system, which has the capability of measuring forces in the range of μN. We tested our system with 40 human subjects to evaluate the role of force feedback in cell injection task. Our experimental results indicate that the subjects were able to feel the cell injection force and confirmed our research hypothesis that the use of combined vision and force feedback leads to higher success rate in cell injection task compared to using vision feedback alone

Mechanical Phenotyping of Mouse Embryonic Stem Cells: Increase in Stiffness with Differentiation

International audienceAtomic force microscopy (AFM) has emerged as a promising tool to characterize the mechanical properties of biological materials and cells. In our studies, undifferentiated and early differentiating mouse embryonic stem cells (mESCs) were assessed individually using an AFM system to determine if we could detect changes in their mechanical properties by surface probing. Probes with pyramidal and spherical tips were assessed, as were different analytical models for evaluating the data. The combination of AFM probing with a spherical tip and analysis using the Hertz model provided the best fit to the experimental data obtained and thus provided the best approximation of the elastic modulus. Our results showed that after only 6 days of differentiation, individual cell stiffness increased significantly with early differentiating mESCs having an elastic modulus two- to threefold higher than undifferentiated mESCs, regardless of cell line (R1 or D3 mESCs) or treatment. Singletouch (indentation) probing of individual cells is minimally invasive compared to other techniques. Therefore, this method of mechanical phenotyping should prove to be a valuable tool in the development of improved methods of identification and targeted cellular differentiation of embryonic, adult, and induced-pluripotent stem cells for therapeutic and diagnostic purposes

Atomic force microscopy-based single-cell indentation: Experimentation and finite element simulation

International audienceIn order to understand and characterize the mechanical property and response of the mouse embryonic stem cells (mESC), we used an atomic force microscope (AFM) combined with a PHANToM haptic feedback device. Atomic force microscopy has rapidly become a valuable tool for quantifying the biophysical properties of single cells or a collection of cells through force measurements. We report herein the mechanical characterization of single mESC using indentation-relaxation measurements with micro-sphere AFM probes for fixed and live undifferentiated mESC. During cell indentation for both live and fixed undifferentiated cells, we provided force feedback to the user in real-time through the PHANToM haptic feedback device as the AFM tip was deforming the cell. The force was amplified for the human operator to perceive the change in force during cell indentation by the AFM cantilever. This information can be used as a mechanical marker to characterize state of the cell (live and fixed). As the interpretation of atomic force microscopy-based indentation tests is highly dependent on the use of an appropriate theoretical model of the testing configuration, various contact models are presented to predict the mechanical behavior of an individual mouse embryonic stem cells (mESC) in different states. A comparison study with finite element simulations (FEM) of spherical tip indentation demonstrates the effectiveness of our computational model to predict the mESC deformation during indentation and relaxation nanomanipulation tasks

Reality-Based Real-Time Cell Indentation Simulator

International audienceTraining simulators that provide realistic visual and haptic feedback during cell indentation tasks are currently inves tigated. Complex cell geometry inherent to biological cells and intricate mechanical properties drive the need for precise mechan ical and numerical modeling to assure accurate cell deformation and force calculations. Advances in alternative finite-element for mulation, such as the mass-tensor approach, have reached a state, where they are applicable to model soft-cell deformation in real time. The geometrical characteristics and the mechanical proper ties of different cells are determined with atomic force microscopy (AFM) indentation. A real-time, haptics-enabled simulator for cell centered indentation has been developed, which utilizes the AFM data (mechanical and geometrical properties of embryonic stem cells) to accurately replicate the indentation task and predict the cell deformation during indentation in real time. This tool can be used as a mechanical marker to characterize the biological state of the cell. The operator is able to feel the change in the stiff ness during cell deformation between fixed and live cells in real time. A comparative study with finite-element simulations using a commercial software and the experimental data demonstrate the effectiveness of the proposed physically based model

Orienting Actin Filaments for Directional Motility of Processive Myosin Motors

To utilize molecular motors in manmade systems, it is necessary to control the motors’ motion. We describe a technique to orient actin filaments so that their barbed ends point in the same direction, enabling same-type motors to travel unidirectionally. Myosin-V and myosin-VI were observed to travel, respectively, toward and away from the filaments’ barbed ends. When both motors were present, they occasionally passed each other while “walking” in opposite directions along single actin filaments

Orienting Actin Filaments for Directional Motility of Processive Myosin Motors

To utilize molecular motors in manmade systems, it is necessary to control the motors’ motion. We describe a technique to orient actin filaments so that their barbed ends point in the same direction, enabling same-type motors to travel unidirectionally. Myosin-V and myosin-VI were observed to travel, respectively, toward and away from the filaments’ barbed ends. When both motors were present, they occasionally passed each other while “walking” in opposite directions along single actin filaments