One-Step Seeding of Neural Stem Cells with Vitronectin-Supplemented Medium for High-Throughput Screening Assays

Abstract

Human neuronal cells differentiated from induced pluripotent cells have emerged as a new model system for the study of disease pathophysiology and evaluation of drug efficacy. Differentiated neuronal cells are more similar in genetics and biological content to the human brain cells than other animal disease models. However, culture of neuronal cells in assay plates requires a labor-intensive procedure of plate pre-coating, hampering its applications in high throughput screening (HTS). We developed a simplified method with one-step seeding of neural stem cells in assay plates by supplementing the medium with a recombinant human vitronectin (VTN), thus avoiding plate pre-coating. Robust results were obtained from cell viability, calcium response, and neurite outgrowth assays using this new method. Our data demonstrate that this approach greatly simplifies high throughput assays using neuronal cells differentiated from human stem cells for translational research.

Enrichment of NPC1-deficient cells with the lipid LBPA stimulates autophagy, improves lysosomal function, and reduces cholesterol storage

“…All cells were below passage 15. WT (GM05659) and NPC1 (GM03123) patient skin fibroblasts were differentiated to neural stem cells (NSC) via iPSC reprogramming using standard laboratory techniques (31,98). NSCs were cultured in Matrigel (Corning) coated T-75 flasks at passage 9, and seeded in 96-well PDL-coated plates (Greiner Bio One) using Knockout DMEM (Thermo Fisher Scientific) supplemented with bFGF (Thermo Fisher Scientific), EGF (Thermo Fisher Scientific), StemPro neural supplement (Thermo Fisher Scientific) and Glutamax plus (Thermo Fisher Scientific), 10 µg/mL vitronectin (Thermo Fisher Scientific) and 1:400 dilution of Matrigel at a density of 10,000 (WT) or 12,000 (NPC1) cells per well for experiments.…”

Vascular-derived SPARC and SerpinE1 regulate interneuron tangential migration and accelerate functional maturation of human stem cell-derived interneurons

“…This interferes with its binding to integrin αV, resulting in greater integrin αV-fibronectin binding and reduced cell adhesion. In the context of neural stem cells, the balance between integrin αV interactions with vitronectin and fibronectin helps to regulate self-renewal versus differentiation ( Dai et al, 2016 ; Varun et al, 2017 ). Vitronectin has a similar role in regulating differentiation in cerebellar granule precursors ( Hashimoto et al, 2016 ).…”

Simple and effective serum-free medium for sustained expansion of bovine satellite cells for cell cultured meat

“…Because one priority of this work was media and cell culture workflow simplicity, we focused on recombinant adhesive proteins that were relatively low-cost and which have been demonstrated without precoating [e.g., laminin-511 fragment (iMatrix-511) and truncated vitronectin (Vtn-N)] 17,18 . We also explored Poly-D-Lysine (PDL) coatings (which can be purchased commercially on precoated plates) to augment cell attachment with or without adhesive peptides 18 . The results indicated that delaying the addition of rAlbumin was necessary to allow cell adhesion and growth, as is coating with a cell adhesive peptide such as iMatrix-511 (Fig.…”

“…To overcome these possible limitations, we explored passaging BSCs in the absence of albumin and adding albumin one day after plating, in order to allow cells to adhere to the flasks, as well as passaging cells with various concentrations of different adhesive proteins. Because one priority of this work was media and cell culture workflow simplicity, we focused on recombinant adhesive proteins that were relatively low-cost and which have been demonstrated without precoating [e.g., laminin-511 fragment (iMatrix-511) and truncated vitronectin (Vtn-N)] 17,18 . We also explored Poly-D-Lysine (PDL) coatings (which can be purchased commercially on precoated plates) to augment cell attachment with or without adhesive peptides 18 .…”