Mapping the Mechanome-A Protocol for Simultaneous Live Imaging and Quantitative Analysis of Cell Mechanoadaptation and Ingression.

Mechanomics, the mechanics equivalent of genomics, is a burgeoning field studying mechanical modulation of stem cell behavior and lineage commitment. Analogous to mechanical testing of a living material as it adapts and evolves, mapping of the mechanome necessitates the development of new protocols to assess changes in structure and function in live stem cells as they adapt and differentiate. Previous techniques have relied on imaging of cellular structures in fixed cells and/or live cell imaging of single cells with separate studies of changes in mechanical and biological properties. Here we present two complementary protocols to study mechanobiology and mechanoadaptation of live stem cells in adherent and motile contexts. First, we developed and tested live imaging protocols for simultaneous visualization and tracking of actin and tubulin mechanoadaptation as well as shape and volume of cells and their nuclei in adherent model embryonic murine mesenchymal stem cells (C3H/10T1/2) and in a neuroblastoma cell line. Then we applied the protocol to enable quantitative study of primary human mesenchymal stem cells in a motile state, e.g., ingression in a three-dimensional, in vitro cell culture model. Together, these protocols enable study of emergent structural mechanoadaptation of the cell's own cytoskeletal machinery while tracking lineage commitment using phenotypic (quantitative morphology measures) and genotypic (e.g., reverse transcription Polymerase Chain Reaction, rtPCR) methods. These tools are expected to facilitate the mapping of the mechanome and incipient mechanistic understanding of stem cell mechanobiology, from the cellular to the tissue and organ length scales.

Scale-up of nature's tissue weaving algorithms to engineer advanced functional materials.

We are literally the stuff from which our tissue fabrics and their fibers are woven and spun. The arrangement of collagen, elastin and other structural proteins in space and time embodies our tissues and organs with amazing resilience and multifunctional smart properties. For example, the periosteum, a soft tissue sleeve that envelops all nonarticular bony surfaces of the body, comprises an inherently "smart" material that gives hard bones added strength under high impact loads. Yet a paucity of scalable bottom-up approaches stymies the harnessing of smart tissues' biological, mechanical and organizational detail to create advanced functional materials. Here, a novel approach is established to scale up the multidimensional fiber patterns of natural soft tissue weaves for rapid prototyping of advanced functional materials. First second harmonic generation and two-photon excitation microscopy is used to map the microscopic three-dimensional (3D) alignment, composition and distribution of the collagen and elastin fibers of periosteum, the soft tissue sheath bounding all nonarticular bone surfaces in our bodies. Then, using engineering rendering software to scale up this natural tissue fabric, as well as multidimensional weaving algorithms, macroscopic tissue prototypes are created using a computer-controlled jacquard loom. The capacity to prototype scaled up architectures of natural fabrics provides a new avenue to create advanced functional materials.