Controlling fluid flow to improve cell seeding uniformity

Reynolds, P. M. , Holzmann Rasmussen, C., Hansson, M., Dufva, M., Riehle, M. O. and Gadegaard, N. (2018) Controlling fluid flow to improve cell seeding uniformity. PLoS ONE, 13(11), e0207211. (doi:10.1371/journal.pone.0207211) (PMID:30440053) (PMCID:PMC6237340)

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Abstract

Standard methods for seeding monolayer cell cultures in a multiwell plate or dish do not uniformly distribute cells on the surface. With traditional methods, users find aggregation around the circumference, in the centre, or a combination of the two. This variation is introduced due to the macro scale flow of the cell seeding suspension, and movement of the dish before cells can settle and attach to the surface. Reproducibility between labs, users, and experiments is hampered by this variability in cell seeding. We present a simple method for uniform and user-independent the cell seeding using an easily produced uniform cell seeder (UCS) device. This allows precise control of cell density in a reproducible manner. By containing the cell seeding suspension in a defined volume above the culture surface with the UCS, fluctuations in cell density are minimised. Seeding accuracy, as defined by the actual cell density versus the target seeding density is improved dramatically across users with various levels of expertise. We go on to demonstrate the impact of local variation in cell density on the lineage commitment of human embryonic stem cells (hESCs) towards pancreatic endoderm (PE). Variations in the differentiation profile of cells across a culture well closely mirror variations in cell density introduced by seeding method–with the UCS correcting variations in differentiation efficiency. The UCS device provides a simple and reproducible method for uniform seeding across multiple culture systems.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Gadegaard, Professor Nikolaj and Riehle, Dr Mathis and Reynolds, Dr Paul
Authors: Reynolds, P. M., Holzmann Rasmussen, C., Hansson, M., Dufva, M., Riehle, M. O., and Gadegaard, N.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
College of Science and Engineering > School of Engineering > Biomedical Engineering
College of Science and Engineering > School of Engineering > James Watt Nanofabrication Centre
Journal Name:PLoS ONE
Publisher:Public Library of Science
ISSN:1932-6203
ISSN (Online):1932-6203
Copyright Holders:Copyright © 2018 Reynolds et al.
First Published:First published in PLoS ONE 13(11): e0207211
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
470563DTC in cell and proteomic technologies (continuation)Jonathan CooperEngineering and Physical Sciences Research Council (EPSRC)EP/F500424/1ENG - BIOMEDICAL ENGINEERING
600931Development of nanopatterned substrates for the delivery of high quality stem cellsNikolaj GadegaardBiotechnology and Biological Sciences Research Council (BBSRC)BB/K011235/1ENG - BIOMEDICAL ENGINEERING