Fig 1.
Improving the uniformity of cell seeding.
(A) Uniform spacing between each cell is the targeted distribution of cells in a dish when seeding to form a monolayer. The reality is that the same number of cells aggregate around the well edge and well centre resulting in a heterogeneous density distribution. Dashed outline shows example of a surface such as a coverslip in the well, which would often not be centred and suffer from further uneven distribution. Movement of the culture vessel to the incubator causes further redistribution of cells due to fluid flow, ultimately resulting in variation in cell phenotype depending on local changes in density. (B) The uniform cell seeder (UCS) device can be used to control the cell suspension, and therefore cell density, across a surface. A capping lid (blue) above the culture surface (grey) holds the cell suspension (pink) at a uniform height across the area (arrows), which evenly distributes the cell population. (C) The UCS device is filled by pipetting the cell suspension through an inlet. In this case, a 22mm coverslip in a 24 well plate is seeded with a uniform distribution of cells. Through capillary force, the cell suspension is held between the UCS and culture surface. (D) Nuclear staining of cells seeded in a 24-well plate without (left) and with (right) the UCS device after 2 weeks culture. Some aggregation around the edges remains, but the uniformity across the well is significantly improved.
Fig 2.
The UCS corrects seeding uniformity.
(A) Six users of varying experience were tasked with seeding cells in a 12-well plate as evenly as possible at high (400 cells/mm2), medium (200 cells/mm2), and low (400 cells/mm2) densities. Cell number across the well centre is shown for each user without (left) and with (right) the UCS device. The y-axis shows the cell number normalised to the target density for each dataset indicated as a dashed line. (B) & (C) Histograms show the local cell density for all users (n = 6) across the entire well after seeding with a target density of 400 cells/mm2 (indicated by red dashed line). Insets show heatmaps of n = 6 samples seeded by each method. Mean Absolute Error (MAE) is shown for each method. Scale bar: 5mm.
Fig 3.
hESC differentiation mirrors seeding density across a well and can be controlled by the UCS device.
(A) hESCs seeded with (top) and without (bottom) the UCS device at three seeding densities–low (1000 cells/mm2), medium (2000 cells/mm2) and high (3000 cells/mm2). A full 15x15 image array of a 12-well plate diameter is shown. PDX1 positive cells (red) and DAPI (blue). Scale bar: 2mm. (B) cross section montage of PDX1 labelled cells for each condition, as outlined in dashed white box in (A). Seeding cells without the UCS device (labelled -UCS) results in central regions with higher expression of PDX1 whereas PDX1 expression is more evenly distributed where cells were seeded in an even manner. Scale bars: A = 1mm, B = 0.5mm. (D) Quantification of total PDX1 cluster area across the centre of the culture well, measuring average size of PDX1 positive clusters in across the centre (n = 6, bars 95% CI).