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.
Highlights
Experiments involving cell culture, from biomaterial testing[1,2,3] to drug discovery[4] often begin with cells seeded onto a flat surface to form a two dimensional culture
We propose a method of cell seeding which makes use of a simple lid above the culture surface to confine the cell suspension and maintain uniform seeding volume per unit area
This uniform cell seeder (UCS) device has only two strict design parameters: 1) the upper surface is flat and rigid, and 2) it is close enough to the target surface to efficiently wick the suspension into the cavity and hold it by capillary force (Fig 1B)
Summary
Experiments involving cell culture, from biomaterial testing[1,2,3] to drug discovery[4] often begin with cells seeded onto a flat surface to form a two dimensional culture. This is the foundation on which the experiment as a whole is built and is arguably one of the most critical steps. The form of salaries for authors CHR and MH, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section
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