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Static and dynamic 3D culture of neural precursor cells on macroporous cryogel microcarriers
Neural precursor cells have been much studied to further our understanding of the far-reaching and controversial question of adult neurogenesis. Currently, differentiation of primary neural precursor cells from the mouse dentate gyrus via 2-dimentional in vitro culture yields low numbers of neurons, a major hindrance to the field of study. 3-dimentional “neurosphere” culture allows better 3D cell-cell contact, but control over cell differentiation is poor because nutrition and oxygen restrictions at the core of the sphere causes spontaneous differentiation, predominantly to glial cells, not neurons. Our group has developed macroporous scaffolds, which overcome the above-mentioned problems, allowing long-term culture of neural stem cells, which can be differentiated into a much higher yield of neurons. Herein we describe a method for culturing neural precursor cells on RGD peptide functionalized-heparin containing cryogel scaffolds, either in standard non-adherent well-plates (static culture) or in spinner flasks (dynamic culture). This method includes: • The synthesis and characterization of heparin based microcarriers. • A “static” 3D culture method for that does not require spinner flask equipment. • “Dynamic” culture in which cell loaded microcarriers are transferred to a spinner flask. © 2020 The Authors
Screening Arrays of Laminin Peptides on Modified Cellulose for Promotion of Adhesion of Primary Endothelial and Neural Precursor Cells
Neural precursor cells (NPC) are primary cells intensively used in the context of research on adult neurogenesis and modeling of neuronal development in health and diseased states. Substrates that can facilitate NPC adhesion will be very useful for culturing these cells. Due to the presence of laminin in basal lamina as well as their involvement in differentiation, migration, and adhesion of many types of cells, surfaces modified with laminin-derived peptides are focused upon and compared with the widely used fibronectin-derived Arg-Gly-Asp (RGD) peptides. An array of 46 peptides is synthesized on cellulose paper (SPOT) to identify laminin-derived peptides that promote short-term adhesion of murine NPC and human primary endothelial cells. Various previously reported peptide sequences are re-evaluated in this work. Initial adhesion experiments show NPC preferred several laminin-derived peptides by up to 5-time higher cell numbers, compared to the well-known promiscuous integrin binding RGD peptide. Importantly, screening of cell adhesion has revealed a synergetic effect of filamentous matrix, peptide sequence, surface property, ligand density, and the dynamic process of NPC adhesion. © The Authors. Advanced Biology published by Wiley-VCH GmbH
Defined Geldrop Cultures Maintain Neural Precursor Cells
Distinct micro-environmental properties have been reported to be essential for maintenance of neural precursor cells (NPCs) within the adult brain. Due to high complexity and technical limitations, the natural niche can barely be studied systematically in vivo. By reconstituting selected environmental properties (adhesiveness, proteolytic degradability, and elasticity) in geldrop cultures, we show that NPCs can be maintained stably at high density over an extended period of time (up to 8 days). In both conventional systems, neurospheres and monolayer cultures, they would expand and (in the case of neurospheres) differentiate rapidly. Further, we report a critical dualism between matrix adhesiveness and degradability. Only if both features are functional NPCs stay proliferative. Lastly, Rho-associated protein kinase was identified as part of a pivotal intracellular signaling cascade controlling cell morphology in response to environmental cues inside geldrop cultures. Our findings demonstrate that simple manipulations of the microenvironment in vitro result in an important preservation of stemness features in the cultured precursor cells.