Haijiao Liu

Perfusable branching microvessel bed for vascularization of engineered tissues

Vascularization is critical for the survival of engineered tissues in vitro and in vivo. In vivo, angiogenesis involves endothelial cell proliferation and sprouting followed by connection of extended cellular processes and subsequent lumen propagation through vacuole fusion. We mimicked this process in engineering an organized capillary network anchored by an artery and a vein. The network was generated by inducing directed capillary sprouting from vascular explants on micropatterned substrates containing thymosin β4-hydrogel. The capillary outgrowths connected between the parent explants by day 21, a process that was accelerated to 14 d by application of soluble VEGF and hepatocyte growth factor. Confocal microscopy and transmission electron microscopy indicated the presence of tubules with lumens formed by endothelial cells expressing CD31, VE-cadherin, and von Willebrand factor. Cardiac tissues engineered around the resulting vasculature exhibited improved functional properties, cell striations, and cell–cell junctions compared with tissues without prevascularization. This approach uniquely allows easy removal of the vasculature from the microfabricated substrate and easy seeding of the tissue specific cell types in the parenchymal space.

In Situ Mechanical Characterization of the Cell Nucleus by Atomic Force Microscopy

The study of nuclear mechanical properties can provide insights into nuclear dynamics and its role in cellular mechanotransduction. While several methods have been developed to characterize nuclear mechanical properties, direct intracellular probing of the nucleus in situ is challenging. Here, a modified AFM (atomic force microscopy) needle penetration technique is demonstrated to mechanically characterize cell nuclei in situ. Cytoplasmic and nuclear stiffness were determined based on two different segments on the AFM indentation curves and were correlated with simultaneous confocal Z-stack microscopy reconstructions. On the basis of direct intracellular measurement, we show that the isolated nuclei from fibroblast-like cells exhibited significantly lower Youngs moduli than intact nuclei in situ. We also show that there is in situ nucleus softening in the highly metastatic bladder cancer cell line T24 when compared to its less metastatic counterpart RT4. This technique has potential to become a reliable quantitative measurement tool for intracellular mechanics studies.

Microfabricated perfusable cardiac biowire: a platform that mimics native cardiac bundle

Tissue engineering enables the generation of three-dimensional (3D) functional cardiac tissue for pre-clinical testing in vitro, which is critical for new drug development. However, current tissue engineering methods poorly recapitulate the architecture of oriented cardiac bundles with supporting capillaries. In this study, we designed a microfabricated bioreactor to generate 3D micro-tissues, termed biowires, using both primary neonatal rat cardiomyocytes and human embryonic stem cell (hESC) derived cardiomyocytes. Perfusable cardiac biowires were generated with polytetrafluoroethylene (PTFE) tubing template, and were integrated with electrical field stimulation using carbon rod electrodes. To demonstrate the feasibility of this platform for pharmaceutical testing, nitric oxide (NO) was released from perfused sodium nitroprusside (SNP) solution and diffused through the tubing. The NO treatment slowed down the spontaneous beating of cardiac biowires based on hESC derived cardiomyocytes and degraded the myofibrillar cytoskeleton of the cardiomyocytes within the biowires. The biowires were also integrated with electrical stimulation using carbon rod electrodes to further improve phenotype of cardiomyocytes, as indicated by organized contractile apparatus, higher Youngs modulus, and improved electrical properties. This microfabricated platform provides a unique opportunity to assess pharmacological effects on cardiac tissue in vitro by perfusion in a cardiac bundle model, which could provide improved physiological relevance.

Determination of local and global elastic moduli of valve interstitial cells cultured on soft substrates

The elasticity of the extracellular matrix profoundly affects biological responses of cells, but also their mechanical properties. Single cell mechanical properties are often measured by atomic force microscopy (AFM), but technical guidelines for AFM measurement of cells grown on soft substrates are not well established. In this study, the local and global elastic moduli of aortic valve interstitial cells (VICs) cultured on soft polyacrylamide substrates (3-144kPa) were characterized via AFM force mapping using pyramidal and spherical tips, respectively. Local and global VIC modulus values both increased with substrate stiffness (p<0.05), with the average local cell modulus being consistently two to three times greater than the global modulus (p<0.05). For local measurements, a minimum of four measurements was required to observe the trend of increasing cell modulus with substrate stiffness, but there was no advantage to testing additional spots. The Hertz model was relatively accurate in estimating the global cell elastic modulus (<12% error, based on validated finite element analyses), despite the cells being of finite thickness and grown on deformable substrates, neither of which are accounted for in the Hertz model. The results of this study provide practical guidelines for efficient AFM-based measurement of the mechanical properties of cells on gels. They also provide new physiologically-relevant data on VIC mechanical properties and their correlation with substrate stiffness-dependent cytoskeletal changes, with relevance to heart valve mechanobiology and disease.

Biophysical Characterization of Bladder Cancer Cells with Different Metastatic Potential

Specific membrane capacitance (SMC) and Young’s modulus are two important parameters characterizing the biophysical properties of a cell. In this work, the SMC and Young’s modulus of two cell lines, RT4 and T24, corresponding to well differentiated (low grade) and poorly differentiated (high grade) urothelial cell carcinoma (UCC), respectively, were quantified using microfluidic and AFM measurements. Quantitative differences in SMC and Young’s modulus values of the high-grade and low-grade UCC cells are, for the first time, reported.

A microfabricated platform with hydrogel arrays for 3D mechanical stimulation of cells.

UNLABELLED Cellular microenvironments present cells with multiple stimuli, including not only soluble biochemical and insoluble matrix cues but also mechanical factors. Biomaterial array platforms have been used to combinatorially and efficiently probe and define two-dimensional (2D) and 3D microenvironmental cues to guide cell functions for tissue engineering applications. However, there are few examples of array platforms that include dynamic mechanical forces, particularly to enable stretching of 3D cell-seeded biomaterials, which is relevant to engineering connective and cardiovascular tissues. Here we present a deformable membrane platform that enables 3D dynamic mechanical stretch of arrayed biomaterial constructs. Cell-seeded polyethylene glycol norbornene (PEG-NB) hydrogels were bound to miniaturized deformable membranes via a thiol-ene reaction with off-stoichiometry thiol-ene based polydimethylsiloxane (OSTE-PDMS) as the membrane material. Bonding to OSTE-PDMS enabled the 3D hydrogel microconstructs to be cyclically deformed and stretched by the membrane. As a first demonstration, human mesenchymal stromal cells (MSCs) embedded in PEG-NB were stretched for several days. They were found to be viable, spread in the 3D hydrogels, and exhibited a contractile myofibroblast phenotype when exposed to dynamic 3D mechanical deformation. This platform, which is readily scalable to larger arrays, enables systematic interrogation of the relationships between combinations of 3D mechanobiological cues and cellular responses, and thus has the potential to identify strategies to predictably control the construction of functional engineered tissues. STATEMENT OF SIGNIFICANCE Current high-throughput biomaterial screening approaches fail to consider the effects of dynamic mechanical stimulation, despite its importance in a wide variety of regenerative medicine applications. To meet this need, we developed a deformable membrane platform that enables 3D dynamic stretch of arrayed biomaterial constructs. Our approach combines microtechnologies fabricated with off-stoichiometry thiol-ene based polydimethylsiloxane membranes that can covalently bond cell-seeded polyethylene glycol norbornene 3D hydrogels, a model biomaterial with tunable adhesive, elastic and degradation characteristics. As a first demonstration, we show that human mesenchymal stromal cells embedded in hydrogels and subjected to dynamic mechanical stimulation undergo myofibroblast differentiation. This system is readily scaled up to larger arrays, and will enable systematic and efficient screening of combinations of 3D mechanobiological and biomaterial cues on cell fate and function.

Automated Vitrification of Embryos: A Robotics Approach

This article reports on the first robotic system for vitrification of mammalian embryos. Vitrification is a technique used for preserving oocytes and embryos in clinical in vitro fertilization (IVF). The procedure involves multiple steps of stringently timed pick-and-place operation for processing an oocyte/embryo in vitrification media. In IVF clinics, the vitrification is conducted manually by highly skilled embryologists. Processing one oocyte/embryo takes the embryologist 15-20 min, depending on the protocols chosen to implement. Due to poor reproducibility and inconsistency across operators, the success and survival rates also vary significantly. Through collaboration with IVF clinics, we are in the process of realizing robotic vitrification (RoboVitri) and ultimately aim to standardize clinical vitrification from manual operation to fully automated robotic operation. Our robotic system is embedded with two contact detection methods to determine the relative z positions of the vitrification micropipette, embryo, and vitrification straw. A three-dimensional (3-D) tracking algorithm is developed for visually served embryo transfer and real-time monitoring of embryo volume changes during vitrification. The excess medium is automatically removed from around the vitrified embryo on the vitrification straw to achieve a high cooling rate.

Cell and Tissue Scale Forces Coregulate Fgfr2-Dependent Tetrads and Rosettes in the Mouse Embryo

What motivates animal cells to intercalate is a longstanding question that is fundamental to morphogenesis. A basic mode of cell rearrangement involves dynamic multicellular structures called tetrads and rosettes. The contribution of cell-intrinsic and tissue-scale forces to the formation and resolution of these structures remains unclear, especially in vertebrates. Here, we show that Fgfr2 regulates both the formation and resolution of tetrads and rosettes in the mouse embryo, possibly in part by spatially restricting atypical protein kinase C, a negative regulator of non-muscle myosin IIB. We employ micropipette aspiration to show that anisotropic tension is sufficient to rescue the resolution, but not the formation, of tetrads and rosettes in Fgfr2 mutant limb-bud ectoderm. The findings underscore the importance of cell contractility and tissue stress to multicellular vertex formation and resolution, respectively.

Polyacrylamide gel substrates that simulate the mechanical stiffness of normal and malignant neuronal tissues increase protoporphyin IX synthesis in glioma cells

Abstract. Protoporphyrin IX (PPIX) produced following the administration of exogenous 5d-aminolevulinic acid is clinically approved for photodynamic therapy and fluorescence-guided resection in various jurisdictions around the world. For both applications, quantification of PPIX forms the basis for accurate therapeutic dose calculation and identification of malignant tissues for resection. While it is well established that the PPIX synthesis and accumulation rates are subject to the cell’s biochemical microenvironment, the effect of the physical microenvironment, such as matrix stiffness, has received little attention to date. Here we studied the proliferation rate and PPIX synthesis and accumulation in two glioma cell lines U373 and U118 cultured under five different substrate conditions, including the conventional tissue culture plastic and polyacrylamide gels that simulated tissue stiffness of normal brain (1 kPa) and glioblastoma tumors (12 kPa). We found that the proliferation rate increased with substrate stiffness for both cell lines, but not in a linear fashion. PPIX concentration was significantly higher in cells cultured on tissue-simulating gels than on the much stiffer tissue culture plastic for both cell lines. These findings, albeit preliminary, suggest that the physical microenvironment might be an important determinant of tumor aggressiveness and PPIX synthesis in glioma cells.

Mechanical stability of the cell nucleus: roles played by the cytoskeleton in nuclear deformation and strain recovery

ABSTRACT Extracellular forces transmitted through the cytoskeleton can deform the cell nucleus. Large nuclear deformations increase the risk of disrupting the integrity of the nuclear envelope and causing DNA damage. The mechanical stability of the nucleus defines its capability to maintain nuclear shape by minimizing nuclear deformation and allowing strain to be minimized when deformed. Understanding the deformation and recovery behavior of the nucleus requires characterization of nuclear viscoelastic properties. Here, we quantified the decoupled viscoelastic parameters of the cell membrane, cytoskeleton, and the nucleus. The results indicate that the cytoskeleton enhances nuclear mechanical stability by lowering the effective deformability of the nucleus while maintaining nuclear sensitivity to mechanical stimuli. Additionally, the cytoskeleton decreases the strain energy release rate of the nucleus and might thus prevent shape change-induced structural damage to chromatin. Summary: The viscoelastic parameters of the cell membrane, cytoskeleton and nucleus are decoupled, and the roles played by cytoskeleton in maintaining nuclear mechanical stability are deciphered.