Cell culture - why uneven distribution of monolayer?

Hello Anika Dreier

You need to consider the following.

1. Inadequate mixing of culture media during addition to the vessel can result in uneven distribution, attachment, and growth of cells. Adding the media and gently tilting the vessel in an appropriate manner will result in an effective way of mixing medium thoroughly without creating bubbles or foam.

2. Uneven growth can also occur because of the shear forces generated by medium sweeping across the cell monolayer during medium changes or while moving cultures between the laminar flow hood and the incubator.

3. You should also consider the factor of levelness. Unusual patterns occur when the vessel in the incubator is not level. Improper stacking of vessels, or if you use shelves in the incubator that have not been leveled prior to use may often cause this effect. The shelves in the incubator should be checked with a spirit level and adjustments should be made following the incubator manufacturer’s recommendations. You should periodically check and make sure that the shelves are level, especially when they are removed for cleaning.

4. You may also get an unusual growth pattern caused by an incubator vibration problem.

5. Temperature differences within the incubator, even though may sound trivial, may create such a problem even when the differences are only a few tenths of a degree. For instance, cells prefer the warmer areas which are directly over the metal portion of the shelf. These conditions usually occur when incubators are frequently opened, especially during the first few hours after freshly inoculated cultures are placed inside the incubator. On the other hand, if the temperature in the incubator is a bit too high for the cells, then the cells would prefer the holes in the shelf for cell growth and attachment. This clearly shows that cells are sensitive to very small temperature changes.

6. While performing cell culture, if you practice stacking vessels together in case of lack of space in the incubator, it can also result in vessel-to-vessel differences in temperature and growth rate. The vessel placed at the bottom of the stack, which is in contact with the metal shelf, warms up the fastest when initially placed in the incubator. The vessel on the top is likely to cool faster, while the vessel in the middle is more insulated from any temperature fluctuations. It is important that you consider these positional effects when you are placing your cells in the incubator.

Hope these may help!

Best.

Saif Wahid

There could be several reasons for the uneven distribution of cells in your monolayer. Here are some possible causes:

Incorrect cell seeding density: If the cell seeding density is too high, it can lead to overcrowding, which can cause cells to detach from the surface and float away from the area where they were initially seeded. On the other hand, if the seeding density is too low, it can result in areas with sparse cell growth. Make sure to refer to the recommended seeding density for your specific cell type and adjust accordingly.

Poor attachment of cells to the substrate: Fish cells require a specialized substrate to attach and grow. Ensure that your flasks are coated with an appropriate extracellular matrix (ECM) that supports the attachment and growth of fish cells. You can try adding a layer of ECM to the flask before seeding the cells.

Insufficient nutrient supply: Cells require a constant supply of nutrients to grow and divide. Check that your medium composition provides adequate nutrients, including glucose, amino acids, vitamins, and minerals. Consider supplementing your medium with additional nutrients, such as serum or growth factors, to support cell growth.

Temperature fluctuations: Temperature affects cell growth rates and metabolism. Fluctuations in temperature can stress cells and alter their growth patterns. Maintain a consistent temperature throughout the incubation period, ideally between 20-25°C for most fish cell lines.

Inadequate gas exchange: Cells require oxygen and release carbon dioxide during metabolic processes. Ensure that your incubator has sufficient gas exchange capabilities to maintain optimal O2 levels and remove CO2. High CO2 concentrations can inhibit cell growth and cause cell death.

Contamination: Contamination Bacteria, fungi, or viruses can contaminate your cultures and cause uneven cell growth. Strictly adhere to sterile technique when handling cells and media, and regularly monitor for signs of contamination.

Genetic drift: As cells divide and expand, genetic mutations can occur randomly. Over time, these mutations can accumulate and result in changes to cell morphology, growth rates, and behavior. Regularly passaging cells through fresh medium can minimize the effects of genetic drift.

Environmental influences: External factors, such as exposure to light, electromagnetic fields, or vibrations, can influence cell growth patterns. Minimize exposure to external stimuli by placing your incubator in a stable environment with minimal disturbances.

Cell-cell contact inhibition: When cells reach confluence, they can exhibit contact inhibition, which slows down or stops cell division. To address this, try sub-culturing cells at regular intervals to maintain a healthy population density.

Heterogeneous cell populations: Your cell line may contain heterogeneous cell populations with varying growth rates, morphologies, or properties. This diversity can lead to uneven cell distribution in the monolayer. Use techniques like flow cytometry or microscopy to analyze your cell population and identify potential differences.

To improve the uniformity of cell growth in your monolayer, consider implementing the following strategies:

Seeding density optimization: Adjust the initial cell seeding density based on the recommendations for your specific cell type. Perform experiments with different seeding densities to determine the ideal concentration for your system.

Enhanced nutrient supply: Supplement your medium with growth factors, hormones, or other nutrients that promote cell growth and attachment.

Improved gas exchange: Ensure proper functioning of your incubator's gas exchange mechanism and maintain optimal atmospheric conditions (O2, CO2, humidity) for your cell type.

Reduced environmental stress: Minimize exposure to external stimuli, such as light, noise, or vibrations, and maintain a stable incubator environment.

Regular passaging: Sub-culture cells at regular intervals to prevent overcrowding and maintain a healthy population density.

Monitoring cell growth and morphology: Regularly inspect your cells using microscopy or other techniques to detect early signs of uneven growth or cell detachment. Adjust your protocols promptly in response to any observed issues.

Contamination control: Strictly adhere to sterile technique and perform routine checks for contamination to minimize the risk of bacterial,

All the best

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