Insights into cell seeding and the tea leaf phenomenon
Proper cell culture seeding is crucial for successful experiments that involve testing for cellular reactivity to determine toxic potential. This is particularly important because the outcome of the study (pass/fail) includes examining the cells under the microscope and evaluating the cellular morphology and confluency; this can be assessed if an appropriate cell density is achieved and there is an overall even distribution of cells in the well during seeding. The method and techniques used for proper cell culture seeding may vary depending on the testing lab; therefore, having a clear understanding of the mechanism behind cell seeding is beneficial.
The first step to proper seeding is depositing an appropriate volume of a defined cell suspension onto a plate. ISO 10993-5 requires cells be resuspended in culture medium at a cell density of 1 x 105 cells/mL per well. An accurate cell density can be measured by a hemocytometer or automated cell counter to perform a cell count; this is paired with a formula to calculate the ratio of cell stock in medium to achieve the required density. This calculation step helps prevent inadequate cell density, which can significantly impact valid cytotoxicity assessments. A high cell density can lead to overcrowding and nutrient depletion. Conversely, low cell density may result in decelerated cell growth and failure to meet the dosing requirement of at least 80% cell confluency.
Once cells are transferred to a cell culture plate, even cell distribution is essential since upon dispensation, cells tend to settle in the middle of the well. Even with the appropriate number of cells deposited, an uneven distribution of cells across a plate can make evaluating a device for toxicity challenging; this is because of the difficulty in discerning the difference between seeding variability and actual percentage of cells affected by toxic substances derived from a device.
The tendency of cells to settle in the middle of the well is consistent with the “tea leaf paradox” observed in fluid dynamics. When stirring a cup of tea, the tea leaves shift toward the center and bottom of the cup as opposed to being dispersed outward in spiral centrifuge. Typically, using a pipette when transferring cell stock generates a rotational flow of the culture medium; thus, a centrifugal force pushes the liquid outward. However, friction slows down the medium near the bottom of the plate, which weakens the centrifugal force. As a result, a secondary circular flow develops, which pushes cells at the bottom of the plate to the center of the well. A technique to reach uniformity of cell density and even distribution within each well to combat the tea leaf phenomenon is to avoid swirling the plates since this can cause the medium to move in a circular motion. Instead, gently shake the plate back and forth and side to side three to five times before placing the cell culture plates in the incubator. This technique helps minimize the rotational movement of the medium.
In conclusion, slight changes in cell culture seeding can have notable impact on cellular behavior and cytotoxicity result interpretation. Comprehending the underlying cause of the “tea leaf paradox” and using the proper tools help promote a uniform cell distribution and compactness. The ability to recognize potential issues that may arise in an experiment particularly related to cell seeding can be achieved through involvement in cell culture coupled with close observation, which ultimately leads to an accurate assessment of biological reactivity.