What causes cell detachment after replating into a monolayer?

08 February 2024 1 9K Report

I dissociated EBs and replated hiPS-CM into a monolayer in a fibronectin coated plates. I had some cells in 96-well plate and others in a 12-well plate. After 24 hrs., cells became adherent and started to contract. However, after 48 hrs., I noticed cells in the 96-well plate started to detach, whereas the cells in 12-well plate were intact. What could be the reason for cells detachment? and how can I avoid this in the future?

Volodymyr Dzhyvak

Dear Alaa Kaoud

The detachment of cells after replating into a monolayer can occur due to various reasons, including:

Inadequate cell adhesion: Despite using fibronectin-coated plates, there might be variations in the quality or quantity of coating between the 96-well plate and the 12-well plate. Insufficient coating or uneven distribution of fibronectin can lead to poor cell adhesion in the 96-well plate, resulting in cell detachment.

Variability in cell density: The cell density at the time of replating can influence cell adhesion and survival. Cells plated at higher densities may experience increased competition for nutrients and space, leading to detachment. Ensure consistent and appropriate cell density across all plates to minimize variability.

Differences in culture conditions: Environmental factors such as temperature, humidity, and CO2 levels can vary between different culture vessels, potentially affecting cell behavior. Make sure to maintain consistent culture conditions across all plates to minimize discrepancies.

Handling and manipulation: Variations in handling and manipulation techniques during replating can impact cell viability and adhesion. Ensure gentle handling and consistent methodology when transferring cells to different plates to minimize stress and damage.

To avoid cell detachment in the future, consider the following strategies:

Standardize coating procedures: Ensure consistent and thorough coating of culture plates with fibronectin to promote uniform cell adhesion. Optimize coating conditions such as concentration, incubation time, and temperature to achieve optimal results.

Maintain consistent cell density: Determine an optimal cell density for replating based on previous experience and experimental requirements. Avoid overcrowding or under-seeding cells to prevent competition-induced detachment.

Monitor culture conditions: Regularly monitor and maintain consistent culture conditions, including temperature, humidity, and CO2 levels, to create a stable environment conducive to cell attachment and growth.

Standardize handling procedures: Establish standardized protocols for cell handling and replating to minimize variability and ensure reproducibility. Train personnel to follow these protocols consistently to maintain experimental consistency.

By implementing these strategies and optimizing experimental conditions, you can minimize the risk of cell detachment and improve the reproducibility of your experiments. Additionally, troubleshooting specific issues encountered during cell culture can help identify and address underlying factors contributing to cell detachment.

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