title: Rotary orbital suspension culture of embryonic stem cell-derived neural stem/progenitor cells: impact of hydrodynamic culture on aggregate yield, morphology and cell phenotype.
abstract: Embryonic stem (ES)-derived neural stem/progenitor cells (ES-NSPCs) constitute a promising cell source for application in cell therapies for the treatment of central nervous system disorders. In this study, a rotary orbital hydrodynamic culture system was applied to single-cell suspensions of ES-NSPCs, to obtain homogeneously-sized ES-NSPC cellular aggregates (neurospheres). Hydrodynamic culture allowed the formation of ES-NSPC neurospheres with a narrower size distribution than statically cultured neurospheres, increasing orbital speeds leading to smaller-sized neurospheres and higher neurosphere yield. Neurospheres formed under hydrodynamic conditions (72 h at 55 rpm) showed higher cell compaction and comparable percentages of viable, dead, apoptotic and proliferative cells. Further characterization of cellular aggregates provided new insights into the effect of hydrodynamic shear on ES-NSPC behaviour. Rotary neurospheres exhibited reduced protein levels of N-cadherin and β-catenin, and higher deposition of laminin (without impacting fibronectin deposition), matrix metalloproteinase-2 (MMP-2) activity and percentage of neuronal cells. In line with the increased MMP-2 activity levels found, hydrodynamically-cultured neurospheres showed higher outward migration on laminin. Moreover, when cultured in a 3D fibrin hydrogel, rotary neurospheres generated an increased percentage of neuronal cells. In conclusion, the application of a constant orbital speed to single-cell suspensions of ES-NSPCs, besides allowing the formation of homogeneously-sized neurospheres, promoted ES-NSPC differentiation and outward migration, possibly by influencing the expression of cell-cell adhesion molecules and the secretion of proteases/extracellular matrix proteins. These findings are important when establishing the culture conditions needed to obtain uniformly-sized ES-NSPC aggregates, either for use in regenerative therapies or in in vitro platforms for biomaterial development or pharmacological screening. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: Instituto de Engenharia Biomédica (INEB), Universidade do Porto, Portugal.|Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Portugal.
authors.forename: Tiago L
authors.initials: TL
authors.lastname: Laundos
authors.affiliation: Instituto de Engenharia Biomédica (INEB), Universidade do Porto, Portugal.|Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Portugal.
authors.forename: Joana
authors.initials: J
authors.lastname: Silva
authors.affiliation: Instituto de Engenharia Biomédica (INEB), Universidade do Porto, Portugal.|Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Portugal.
authors.forename: Marisa
authors.initials: M
authors.lastname: Assunção
authors.affiliation: Instituto de Engenharia Biomédica (INEB), Universidade do Porto, Portugal.|Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Portugal.
authors.forename: Pedro
authors.initials: P
authors.lastname: Quelhas
authors.affiliation: Instituto de Engenharia Biomédica (INEB), Universidade do Porto, Portugal.|Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Portugal.
authors.forename: Cátia
authors.initials: C
authors.lastname: Monteiro
authors.affiliation: Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Portugal.|Expression Regulation in Cancer Group, Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Portugal.|Departamento de Patologia e Oncologia, Faculdade de Medicina, Universidade do Porto, Portugal.
authors.forename: Carla
authors.initials: C
authors.lastname: Oliveira
authors.affiliation: Instituto de Engenharia Biomédica (INEB), Universidade do Porto, Portugal.|Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Portugal.|Departamento de Patologia e Oncologia, Faculdade de Medicina, Universidade do Porto, Portugal.
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Oliveira
authors.affiliation: Instituto de Engenharia Biomédica (INEB), Universidade do Porto, Portugal.|Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Portugal.|Faculdade de Engenharia, Universidade do Porto, Portugal.|Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Portugal.
authors.forename: Ana P
authors.initials: AP
authors.lastname: Pêgo
authors.affiliation: Instituto de Engenharia Biomédica (INEB), Universidade do Porto, Portugal.|Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Portugal.|Faculdade de Engenharia, Universidade do Porto, Portugal.
authors.forename: Isabel F
authors.initials: IF
authors.lastname: Amaral
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002460:Cell Line; D057446:Hydrodynamics; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D058953:Neural Stem Cells / Q000166:cytology / Q000378:metabolism; D018874:Spheroids, Cellular / Q000166:cytology / Q000378:metabolism
keywords: 3D fibrin hydrogel; cell differentiation; cell migration; cell-cell adhesion molecules; extracellular matrix deposition; hydrodynamic culture; matrix metalloproteinases; neural stem cells