Dendritic Polyglycerol Amine: An Enhanced Substrate to Support Long-Term Neural Cell Culture

Long-term stable cell culture is a critical tool to better understand cell function. Most adherent cell culture models require a polymer substrate coating of poly-lysine or poly-ornithine for the cells to adhere and survive. However, polypeptide-based substrates are degraded by proteolysis and it remains a challenge to maintain healthy cell cultures for extended periods of time. Here, we report the development of an enhanced cell culture substrate based on a coating of dendritic polyglycerol amine (dPGA), a non-protein macromolecular biomimetic of poly-lysine, to promote the adhesion and survival of neurons in cell culture. We show that this new polymer coating provides enhanced survival, differentiation and long-term stability for cultures of primary neurons or neurons derived from human induced pluripotent stem cells (hiPSCs). Atomic force microscopy analysis provides evidence that greater nanoscale roughness contributes to the enhanced capacity of dPGA-coated surfaces to support cells in culture. We conclude that dPGA is a cytocompatible, functionally superior, easy to use, low cost and highly stable alternative to poly-cationic polymer cell culture substrate coatings such as poly-lysine and poly-ornithine. Summary statement Here, we describe a novel dendritic polyglycerol amine-based substrate coating, demonstrating superior performance compared to current polymer coatings for long-term culture of primary neurons and neurons derived from induced pluripotent stem cells.

Induction of Anisotropic Orientation and Enhancement of Gene Functional Expression of Human Pluripotent Stem Cell-Derived Cardiomyocytes Cultured on Nanofabricated Substrates Consisting of Micron Planar Lines and Nano Dot Structures.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are expected to have applications in the fields of regenerative medicine and drug discovery. However, the immaturity of hiPS-CMs is an issue to be considered, and in order to replicate in vivo responsivity, there have been several attempts to induce maturation in hiPS-CMs, including methods to induce differentiation of hiPS-CMs by changing culture medium and culture substrate. In particular, anisotropic culture, in which the cultured cells are aligned in one direction, induces the cellular morphology resembling that of in vivo cardiomyocytes and is expected to be a useful method for maturation of hiPS-CMs. We tried forming a nanostructure on the surface of the cell culture substrate using our original nanofabrication technology, with the aim of aligning cardiomyocytes and inducing maturation. Our newly developed nanostructure for anisotropic culture (line/dot structure) comprises a region of cone-shaped nanopillars with pitch distance of several hundred nm and a planar region, alternating in a striped pattern with intervals of several tens of µm, arranged on the surface of the cell culture substrate. The hiPS-CMs cultured using the line/dot structure showed anisotropic orientation, and increased mRNA expression was observed in myocardial structural protein genes, genes relating to ion channels, and the gene for Cx43. These results suggest that the line/dot nanostructure is effective for anisotropic culture and cell maturation of hiPS-CMs.

On the energy efficiency of cell migration in diverse physical environments

In this work, we explore fundamental energy requirements during mammalian cell movement. Starting with the conservation of mass and momentum for the cell cytosol and the actin-network phase, we develop useful identities that compute dissipated energies during extensions of the cell boundary. We analyze 2 complementary mechanisms of cell movement: actin-driven and water-driven. The former mechanism occurs on 2-dimensional cell-culture substrate without appreciable external hydraulic resistance, while the latter mechanism is prominent in confined channels where external hydraulic resistance is high. By considering various forms of energy input and dissipation, we find that the water-driven cell-migration mechanism is inefficient and requires more energy. However, in environments with sufficiently high hydraulic resistance, the efficiency of actin-polymerization-driven cell migration decreases considerably, and the water-based mechanism becomes more efficient. Hence, the most efficient way for cells to move depends on the physical environment. This work can be extended to higher dimensions and has implication for understanding energetics of morphogenesis in early embryonic development and cancer-cell metastasis and provides a physical basis for understanding changing metabolic requirements for cell movement in different conditions.