Why wells are created in circular shape

The holes were temporarily sealed with a Kapton tape. S4 30 , The EFSs can then be calculated by dividing the voltage differences by the distance between respective electrodes. Similar procedure was applied to the simple rectangular channel chip. Alternatively, the inserts can be affixed to the well bottom with the presence of PBS as shown in Fig. PBS, cell culture medium, and cell suspension were infused into the microfluidic chip sequentially.

Thereafter the buffer is replaced by cell culture medium and cell suspension is then inoculated. After overnight culture for cell adhesion and growth, fittings to supply culture medium and for salt bridges containing 1. The EF stimulation setup diagram and a snapshot of the 6-well plate is shown in Fig. The time lapse electrotaxis experiments were carried out on an automated microscope Ti-E, Nikon, Japan Supplementary Fig. A p-value the probability for a true null hypothesis less than 0.

The current density at the bottom of the chamber was simulated for the plain polymeric insert, the smooth polymeric insert with the 3D structure designed to intersect a liquid column by paraboloids, and the layered approximation for the PMMA insert see schematics in Fig. With the liquid column thickness of 0. Without the 3D designed structure, a large portion of the electrical current in red passes through the liquid column instead of passing through the bottom chamber where the cells are located Fig.

With the 3D designed structure, the current lines in red are uniformly distributed in the bottom chamber, indicating that a uniform and directional EF was created Fig. The highly directional EF created by using the insert also suggests that the inserts are suitable not only for cell stimulation but also for electrotaxis studies. A uniform decrease in the electric potential in the bottom chamber can be found in Supplementary Fig. The simulated results are summarised in Table 1. In a plain polymeric insert without the 3D designed structure, the EF is non-uniform and the mean EFS and coefficient of variation CV, defined as the ratio of the standard deviation to the mean , are The double sided tape used in this study is a pressure sensitive adhesive prone to deformation under pressure or stretching.

Effect of slight deformation in the chamber thickness to the EF uniformity was examined by numerical simulations. Supplementary Fig. Although the chamber with a height of 0. The EFSs were measured between adjacent holes parallel to the electric current vector. The mean EFS of all measurement is The measured mean EFS is about This value is also comparable to the 2. Finally, the EFSs along the perpendicular direction to the electric current vector is measured to be 4. While conventional in vitro electrical stimulation devices either sacrifice the culture area to stimulate cells uniformly, or stimulate large areas of cells with non-uniform EF, the polymeric circular insert utilised in this study can provide uniform EF stimulation to large area percentage of cells.

The effective stimulation area is defined as the area of the bottom chamber subtracting the area of where the liquid column resides. The effective stimulation area percentage is the ratio of the effective stimulation area over the total surface area of the TCPS dish. The effective stimulation area percentages using the polymeric insert are listed in comparison to those reported in existing literature, see Table 2.

Thus the cell yields are higher by using polymeric inserts for electrical stimulation. The higher cell yields will greatly benefit subsequent biochemical and molecular biology analysis. See more details in SI-Movie To quantify the cell migration and alignment, two parameters directedness and orientation are used with the following defintion, see schematics in Fig.

For a group of randomly migrating cells, the directedness is zero. For a group of randomly shaped cells, the average orientation is zero. The directedness and orientation of the cells with and without EF stimulation are shown in Fig. This deviation is possibly caused by un-optimised cell culture medium flow rate.

While the shear stress in the polymer insert device and the rectangular channel is of the same order, the medium replenishment takes longer for the circular insert due to its bigger cross-sectional area. Before EF stimulation, cells in both rectangular channels and circular inserts demonstrated random orientation 0. The control cells in both rectangular channels and the circular inserts do not show any alignment.

Detailed cell migration and orientation data is shown in Supplementary Table S1. These results validated the performance of the inserts for electrotaxis experiments comparable to the performance of a rectangular channel.

However, the circular inserts have at least two fold higher effective stimulation percentage in comparison to that of rectangular channels, thus more cell yield can be achieved by using our circular inserts. Moreover, a removable polymeric insert can further aid cell recovery right after the EF stimulation, which can be accomplished by adding a perfluoropolymer-coated layer between the adhesive tape and the insert Alternatively, the removable insert can be fabricated by using polydimethylsiloxane as the insert material.

The silicone rubber can reversibly bond to the TCPS dish with air-tight seal by the clip-on design, similar to those in a transwell insert Supplementary Fig. Establishment of a uniform EF in a circular-shaped microdevice is extremely difficult so the majority of existing EF stimulation devices avoided this issue by using a simple rectangular shaped chamber.

The rectangular configuration requires modification to fit with the commercial labware, and only a small portion of the cell culture dish is used for cell culture, thus limiting the cell yield. By adding a 3D CAD based insert in a circular shaped cell culture chamber, we have demonstrated that a uniform EF can be created in a circular-shaped area by modulating the electrical resistance across the device.

We highlight our key contributions and outlooks below. The effective stimulation area percentage using the insert is at least 2 fold higher than that of existing EF stimulation devices.

The yield of cells and its products can be increased for further biochemical analysis. Mass production of the polymeric insert can be achieved by CNC fabrication, injection molding, or other similar technology. The polymeric insert is useful for adapting electrical stimulation studies in a common laboratory due to the high effective stimulation area percentage and the ease of use. The polymeric insert is applicable for various studies.

For tissue engineering, EF stimulation has been reported to induce synchronously contracting cardiac tissue 52 , 53 , 54 , 55 , Osteoblastic differentiation from mesenchymal stem cells can be promoted under EF stimulation Uniform EF stimulation to circular shaped area could also be useful to stimulate an entire brain slice or tissue slice. How to cite this article : Tsai, H. Uniform electric field generation in circular multi-well culture plates using polymeric inserts.

McCaig, C. Electrical dimensions in cell science. J Cell Sci , — Electric fields, contact guidance and the direction of nerve growth. J Embryol Exp Morphol 94 , — Messerli, M. Biol Bull , 79—92 Levin, M. Morphogenetic fields in embryogenesis, regeneration, and cancer: Non-local control of complex patterning. Biosystems , — Golberg, A.

Sci Rep 5 , Giugni, T. Electric field-induced redistribution and postfield relaxation of epidermal growth factor receptors on A cells. J Cell Biol , — Rochlin, M. Localization of intracellular proteins at acetylcholine receptor clusters induced by electric fields in Xenopus muscle cells. We may request cookies to be set on your device. We use cookies to let us know when you visit our websites, how you interact with us, to enrich your user experience, and to customize your relationship with our website.

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Changes will take effect once you reload the page. Government agencies and nonprofit groups are preparing for difficult months ahead. Brett Walton. Donate to Circle of Blue. Short answer is no of course! But how do we know that? Surprisingly, there are still people who try and argue the Earth is flat, based particularly on things like not being able to see a curved horizon from the shore. Image credit: Bored Panda. The problem is that even when we look at the horizon, the size of the Earth means that the curvature is very very small.

Over 10 km wider than the view above , the curvature would be less than 10m from one side to the other. A eye-tracking study found that at five months of age, before they utter a word or scribble a drawing, infants already show a clear visual preference for contoured lines over straight lines.

Over the last century, researchers on human perception have tried to explain our innate propensity for circular and curvy shapes.

In a study conducted by the Swedish psychologist Helge Lundholm, subjects were asked to draw lines representing a set of emotional adjectives. While angular lines were used to depict adjectives like hard, harsh and cruel, curved lines were the popular choice for adjectives like gentle, quiet and mild.

Typography has been the target of a similar analysis. In a seminal paper published in in Psychological Science, cognitive psychologists Moshe Bar and Maital Neta conducted an experiment in which 14 participants were shown pairs of letters, patterns, and everyday objects, differing only in the curvature of their contour. The results were not completely surprising: participants showed a strong preference for curved items in all categories, particularly when it came to real objects.

To better understand this bias, the same pair of scientists conducted another study one year later, this time by mapping the cognitive response using functional magnetic resonance imaging fMRI. The results were quite conclusive. Sharp-cornered objects caused much greater amygdala activation than rounded objects.