Youhua Tan

Soft fibrin gels promote selection and growth of tumorigenic cells

The identification of stem-cell-like cancer cells through conventional methods that depend on stem-cell markers is often unreliable. We developed a mechanical method of selecting tumourigenic cells by culturing single cancer cells in fibrin matrices of ~100 Pa in stiffness. When cultured within these gels, primary human cancer cells or single cancer cells from mouse or human cancer cell lines grew within a few days into individual round colonies that resembled embryonic stem-cell colonies. Subcutaneous or intravenous injection of 10 or 100 fibrin-cultured cells in syngeneic or severe-combined-immunodeficiency mice led to the formation of solid tumours at the site of injection or at the distant lung organ much more efficiently than control cancer cells selected using conventional surface marker methods or cultured on conventional rigid dishes or on soft gels. Remarkably, as few as 10 such cells were able to survive and form tumours in the lungs of wild-type non-syngeneic mice.

Mechanical Characterization of Human Red Blood Cells Under Different Osmotic Conditions by Robotic Manipulation With Optical Tweezers

The physiological functions of human red blood cells (RBCs) play a crucial role to human health and are greatly influenced by their mechanical properties. Any alteration of the cell mechanics may cause human diseases. The osmotic condition is an important factor to the physiological environment, but its effect on RBCs has been little studied. To investigate this effect, robotic manipulation technology with optical tweezers is utilized in this paper to characterize the mechanical properties of RBCs in different osmotic conditions. The effectiveness of this technology is demonstrated first in the manipulation of microbeads. Then the optical tweezers are used to stretch RBCs to acquire the force-deformation relationships. To extract cell properties from the experimental data, a mechanical model is developed for RBCs in hypotonic conditions by extending our previous work , and the finite element model is utilized for RBCs in isotonic and hypertonic conditions. Through comparing the modeling results to the experimental data, the shear moduli of RBCs in different osmotic solutions are characterized, which shows that the cell stiffness increases with elevated osmolality. Furthermore, the property variation and potential biomedical significance of this study are discussed. In conclusion, this study indicates that the osmotic stress has a significant effect on the cell properties of human RBCs, which may provide insight into the pathology analysis and therapy of some human diseases.

Mechanical Modeling of Biological Cells in Microinjection

Microinjection is an effective technique to introduce foreign materials into a biological cell. Although some semi-automatic and fully-automatic microinjection systems have been developed, a full understanding of the mechanical response of biological cells to injection operation remains deficient. In this paper, a new mechanical model based on membrane theory is proposed. This model establishes a relationship between the injection force and the deformation of biological cells with the quasi-static equilibrium equations, which are solved by the Runge-Kutta numerical method. Based on this model, other mechanical responses can also be inferred, such as the effect of the injector radius, the membrane stress and tension distribution, internal cell pressure, and the deformed cell shape. To verify the proposed model, experiments are performed on microinjection of zebrafish embryos at different developmental stages and medaka embryos at the blastula stage. It is demonstrated that the modeling results agree well with the experimental data, which shows that the proposed model can be used to estimate the mechanical properties of cell biomembranes. (In this paper, biomembrane refers to the membrane-like structures enveloping cells.)

Matrix softness regulates plasticity of tumour-repopulating cells via H3K9 demethylation and Sox2 expression

Tumour-repopulating cells (TRCs) are a self-renewing, tumorigenic subpopulation of cancer cells critical in cancer progression. However, the underlying mechanisms of how TRCs maintain their self-renewing capability remain elusive. Here we show that relatively undifferentiated melanoma TRCs exhibit plasticity in Cdc42-mediated mechanical stiffening, histone 3 lysine residue 9 (H3K9) methylation, Sox2 expression and self-renewal capability. In contrast to differentiated melanoma cells, TRCs have a low level of H3K9 methylation that is unresponsive to matrix stiffness or applied forces. Silencing H3K9 methyltransferase G9a or SUV39h1 elevates the self-renewal capability of differentiated melanoma cells in a Sox2-dependent manner. Mechanistically, H3K9 methylation at the Sox2 promoter region inhibits Sox2 expression that is essential in maintaining self-renewal and tumorigenicity of TRCs both in vitro and in vivo. Taken together, our data suggest that 3D soft-fibrin-matrix-mediated cell softening, H3K9 demethylation and Sox2 gene expression are essential in regulating TRC self-renewal.

Generation of organized germ layers from a single mouse embryonic stem cell

Mammalian inner cell mass cells undergo lineage-specific differentiation into germ layers of endoderm, mesoderm and ectoderm during gastrulation. It has been a long-standing challenge in developmental biology to replicate these organized germ layer patterns in culture. Here we present a method of generating organized germ layers from a single mouse embryonic stem cell cultured in a soft fibrin matrix. Spatial organization of germ layers is regulated by cortical tension of the colony, matrix dimensionality and softness, and cell–cell adhesion. Remarkably, anchorage of the embryoid colony from the 3D matrix to collagen-1-coated 2D substrates of ~1 kPa results in self-organization of all three germ layers: ectoderm on the outside layer, mesoderm in the middle and endoderm at the centre of the colony, reminiscent of generalized gastrulating chordate embryos. These results suggest that mechanical forces via cell–matrix and cell–cell interactions are crucial in spatial organization of germ layers during mammalian gastrulation. This new in vitro method could be used to gain insights on the mechanisms responsible for the regulation of germ layer formation.

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.

Mechanical modeling of red blood cells during optical stretching.

Mechanical properties of red blood cells (RBCs) play an important role in regulating cellular functions. Many recent researches suggest that the cell properties or deformability may be used as a diagnostic indicator for the onset and progression of some human diseases. Although optical stretcher (OS) has emerged as an effective tool to investigate the cell mechanics of RBCs, little is known about the deformation behavior of RBCs in an OS. To address this problem, the mechanical model proposed in our previous work is extended in this paper to describe the mechanical responses of RBCs in the OS. With this model, the mechanical responses, such as the tension distribution, the effect of cell radius, and the deformed cell shapes, can be predicted. It is shown that the results obtained from our mechanical model are in good agreement with the experimental data, which demonstrates the validity of the developed model. Based on the derived model, the mechanical properties of RBCs can be further obtained. In conclusion, this study indicates that the developed mechanical model can be used to predict the deformation responses of RBCs during optical stretching and has potential biomedical applications such as characterizing cell properties and distinguishing abnormal cells from normal ones.

Characterizing Mechanical Properties of Biological Cells by Microinjection

Microinjection has been demonstrated to be an effective technique to introduce foreign materials into biological cells. Despite the advance, whether cell injection can be used to characterize the mechanical properties of cells remains elusive. In this paper, extending the previously developed mechanical model, various constitutive materials are adopted to present the membrane characteristics of cells. To demonstrate the modeling approach and identify the most appropriate constitutive material for a specific biomembrane, finite element analysis (FEA) and experimental tests are carried out. It is shown that the modeling results agree well with those from both FEA and experiments, which demonstrates the validity of the developed approach. Moreover, Yeoh and Cheng materials are found to be the best constitutive materials in representing the deformation behaviors of zebrafish embryos and mouse embryos (or oocytes), respectively. Also, the mechanical properties of zebrafish embryos at different developmental stages and mouse embryos (or oocytes) are characterized.

Biophysical characterization of hematopoietic cells from normal and leukemic sources with distinct primitiveness

This letter reported the biophysical characterization of immunophenotypically distinct hematopoietic cells from normal and leukemic sources, through manipulation with optical tweezers at single cell level. The results show that the percentage of cells that are stretchable and their deformability are significantly higher in the more primitive cell populations. This study provides the evidence that normal and leukemic hematopoietic cell populations with distinct primitiveness exhibit differential biophysical properties. These findings raise a hypothesis that the high deformability may be related to the unique functions and activities of primitive hematopoietic cells.

Force analysis and path planning of the trapped cell in robotic manipulation with optical tweezers

Laser trapping in the near infrared regime is a noninvasive and convenient manipulation tool, which can be utilized as micromanipulator for a large number of biological applications. Increasing demands for both accuracy and efficiency in cell manipulation highlight the need for automation process that integrates robotics and tweezers technologies. In this paper, we propose a robotic manipulation system with optical tweezers, and analyze the force applied on the trapped cell for design of an optimal trapping strategy. The dynamic motion of the cell with consideration of both the trapping and the viscous forces is analyzed, based on which the motion profile of the motorized stage is designed to ensure both safety and efficiency of the cell delivery. A modified A-star algorithm is used for path planning in transporting cells. Experiments are performed on manipulating the yeast cells to demonstrate the effectiveness of the proposed approach.