Two-Photon Polymerized Shape Memory Microfibers: A New Mechanical Characterization Method in Liquid.

Two-photon polymerization (TPP) has been widely used to create 3D micro- and nanoscale scaffolds for biological and mechanobiological studies, which often require the mechanical characterization of the TPP fabricated structures. To satisfy physiological requirements, most of the mechanical characterizations need to be conducted in liquid. However, previous characterizations of TPP fabricated structures were all conducted in air due to the limitation of conventional micro- and nanoscale mechanical testing methods. In this study, we report a new experimental method for testing the mechanical properties of TPP-printed microfibers in liquid. The experiments show that the mechanical behaviors of the microfibers tested in liquid are significantly different from those tested in air. By controlling the TPP writing parameters, the mechanical properties of the microfibers can be tailored over a wide range to meet a variety of mechanobiology applications. In addition, it is found that, in water, the plasticly deformed microfibers can return to their pre-deformed shape after tensile strain is released. The shape recovery time is dependent on the size of microfibers. The experimental method represents a significant advancement in mechanical testing of TPP fabricated structures and may help release the full potential of TPP fabricated 3D tissue scaffold for mechanobiological studies.

A multi-material platform for imaging of single cell-cell junctions under tensile load fabricated with two-photon polymerization.

We previously reported a single-cell adhesion micro tensile tester (SCAµTT) fabricated from IP-S photoresin with two-photon polymerization (TPP) for investigating the mechanics of a single cell-cell junction under defined tensile loading. A major limitation of the platform is the autofluorescence of IP-S, the photoresin for TPP fabrication, which significantly increases background signal and makes fluorescent imaging of stretched cells difficult. In this study, we report the design and fabrication of a new SCAµTT platform that mitigates autofluorescence and demonstrate its capability in imaging a single cell pair as its mutual junction is stretched. By employing a two-material design using IP-S and IP-Visio, a photoresin with reduced autofluorescence, we show a significant reduction in autofluorescence of the platform. Further, by integrating apertures onto the substrate with a gold coating, the influence of autofluorescence on imaging is almost completely mitigated. With this new platform, we demonstrate the ability to image a pair of epithelial cells as they are stretched up to 250% strain, allowing us to observe junction rupture and F-actin retraction while simultaneously recording the accumulation of over 800 kPa of stress in the junction. The platform and methodology presented here can potentially enable detailed investigation of the mechanics of and mechanotransduction in cell-cell junctions and improve the design of other TPP platforms in mechanobiology applications.

Spatially Guided Construction of Multilayered Epidermal Models Recapturing Structural Hierarchy and Cell-Cell Junctions.

A current challenge in three-dimensional (3D) bioprinting of skin equivalents is to recreate the distinct basal and suprabasal layers and to promote their direct interactions. Such a structural arrangement is essential to establish 3D stratified epidermis disease models, such as for the autoimmune skin disease pemphigus vulgaris (PV), which targets the cell-cell junctions at the interface of the basal and suprabasal layers. Inspired by epithelial regeneration in wound healing, we develop a method that combines 3D bioprinting and spatially guided self-reorganization of keratinocytes to recapture the fine structural hierarchy that lies in the deep layers of the epidermis. Here, keratinocyte-laden fibrin hydrogels are bioprinted to create geographical cues, guiding dynamic self-reorganization of cells through collective migration, keratinocyte differentiation and vertical expansion. This process results in a region of self-organized multilayers (SOMs) that contain the basal to suprabasal transition, marked by the expressed levels of different types of keratins that indicate differentiation. Finally, we demonstrate the reconstructed skin tissue as an in vitro platform to study the pathogenic effects of PV and observe a significant difference in cell-cell junction dissociation from PV antibodies in different epidermis layers, indicating their applications in the preclinical test of possible therapies.

Nanosensors for single cell mechanical interrogation.

The occurrence and development of many diseases are accompanied and sometimes dictated by the destruction of biomechanical homeostasis. For instance, cancer cells and normal cells show different cellular mechanical forces phenotypes, as the proliferation and invasion ability of cancer cells is often related to the changes in mechanical force in the tumor. With single cell analysis, variations in mechanics within a cell population can be detected and analyzed, opening new dimensions in the study of cancer. Nanosensor design for interrogation of cell mechanics is an interdisciplinary area bridging over cell biology, mechanics, and micro/nanotechnology. In this tutorial review, we give insight into the background and technical innovation of currently available methods for mechanical analysis of cells. First, we discuss the mechanism of mechanical changes in the development and progression of cancer that shows the feasibility of mechanical sensors in cancer cell detection. Next, we summarize the principle, progress, and essential problems of common technologies for cell force measurement, including single molecule force spectroscopy and elastic substrate-sensors. Following that, we discuss novel micro and nano-scale mechanical sensors and their applications in single cell level biological analysis. At last, we elaborate on the remaining issues and trends of the cellular mechanical sensors.

Characterization of the strain-rate-dependent mechanical response of single cell-cell junctions.

Cell-cell adhesions are often subjected to mechanical strains of different rates and magnitudes in normal tissue function. However, the rate-dependent mechanical behavior of individual cell-cell adhesions has not been fully characterized due to the lack of proper experimental techniques and therefore remains elusive. This is particularly true under large strain conditions, which may potentially lead to cell-cell adhesion dissociation and ultimately tissue fracture. In this study, we designed and fabricated a single-cell adhesion micro tensile tester (SCAµTT) using two-photon polymerization and performed displacement-controlled tensile tests of individual pairs of adherent epithelial cells with a mature cell-cell adhesion. Straining the cytoskeleton-cell adhesion complex system reveals a passive shear-thinning viscoelastic behavior and a rate-dependent active stress-relaxation mechanism mediated by cytoskeleton growth. Under low strain rates, stress relaxation mediated by the cytoskeleton can effectively relax junctional stress buildup and prevent adhesion bond rupture. Cadherin bond dissociation also exhibits rate-dependent strengthening, in which increased strain rate results in elevated stress levels at which cadherin bonds fail. This bond dissociation becomes a synchronized catastrophic event that leads to junction fracture at high strain rates. Even at high strain rates, a single cell-cell junction displays a remarkable tensile strength to sustain a strain as much as 200% before complete junction rupture. Collectively, the platform and the biophysical understandings in this study are expected to build a foundation for the mechanistic investigation of the adaptive viscoelasticity of the cell-cell junction.

High Throughput and Highly Controllable Methods for In Vitro Intracellular Delivery.

In vitro and ex vivo intracellular delivery methods hold the key for releasing the full potential of tissue engineering, drug development, and many other applications. In recent years, there has been significant progress in the design and implementation of intracellular delivery systems capable of delivery at the same scale as viral transfection and bulk electroporation but offering fewer adverse outcomes. This review strives to examine a variety of methods for in vitro and ex vivo intracellular delivery such as flow-through microfluidics, engineered substrates, and automated probe-based systems from the perspective of throughput and control. Special attention is paid to a particularly promising method of electroporation using micro/nanochannel based porous substrates, which expose small patches of cell membrane to permeabilizing electric field. Porous substrate electroporation parameters discussed include system design, cells and cargos used, transfection efficiency and cell viability, and the electric field and its effects on molecular transport. The review concludes with discussion of potential new innovations which can arise from specific aspects of porous substrate-based electroporation platforms and high throughput, high control methods in general.