Controlling fluid flow to improve cell seeding uniformity

Paul M. Reynolds*, Camilla Holzmann Rasmussen, Mattias Hansson, Martin Dufva, Mathis O. Riehle, Nikolaj Gadegaard

*Corresponding author for this work

    Research output: Contribution to journalJournal articleResearchpeer-review

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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.

    Original languageEnglish
    Article numbere0207211
    JournalPLOS ONE
    Volume13
    Issue number11
    Number of pages12
    ISSN1932-6203
    DOIs
    Publication statusPublished - 2018

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