Why can't Caco-2 cells reattach to T75 flasks?

Experience About Caco-2 Cell Culture and Passaging

Regarding Cell Condition and Attachment Caco-2 cells inherently attach slowly, especially immediately after thawing or at the early stages of passaging. Their attachment time is generally longer compared to many other common cell lines. Therefore, during routine culture, it is advisable not to observe the cells under the microscope too frequently. Minimizing unnecessary disturbance is actually more beneficial for the cells. Excessive observation and movement of culture flasks may induce the generation of floating cells.

Regarding Serum Concentration The recommended serum concentration in the culture medium for Caco-2 cells is generally around 20%. This concentration is optimal for both cell attachment and growth. If serum concentration is too low, cells do not adhere well and grow slowly; if too high, it may adversely affect their differentiation process. Thus, if cell condition deteriorates, adjusting the serum concentration should be the first consideration.

Causes and Management of Floating Cells If the number of floating cells increases over time, and clusters of cells are observed detaching, this is a warning sign. It is recommended to investigate several factors: (1) Culture medium components, especially the batch and quality of serum, as Caco-2 cells are highly sensitive to serum variations; (2) Culture conditions, including CO2 concentration, humidity, and temperature in the incubator, which must remain stable.

“Pitfalls” in Digestion and Passaging Trypsinization during passaging is a critical and often challenging step. Caco-2 cells form tight intercellular junctions and adhere strongly to the flask surface, making them difficult to digest. The longer the culture duration, the stronger these connections become, increasing digestion difficulty. Generally, a digestion time of 5–10 minutes is recommended, but this depends on cell density and days in culture. It is very difficult to fully dissociate Caco-2 cells into single cells; the goal is to obtain small cell clusters. If some cells have detached but others remain firmly attached, collect the detached cells first by centrifugation, then add fresh trypsin to the remaining cells and return the flask to the incubator for continued digestion. It is advisable to check every minute to avoid over-digestion.

Subtle Tips for Culture Environment Caco-2 cells are sensitive to external disturbances. For example, placing culture flasks near the incubator door or frequently opening and closing the incubator can negatively affect cell status. Therefore, it is best to position culture flasks deeper inside the incubator in a stable location to minimize environmental fluctuations.

The answer to this question comes from MedChemExpress (MCE) Technical Support. [email protected]

Maisra Azhar

A sudden loss of Caco-2 cells after medium change and centrifugation on Day 3 may be due to multiple factors, particularly mechanical stress or suboptimal handling conditions. Caco-2 cells are epithelial in nature and highly sensitive to detachment, especially in early culture stages when cell–cell junctions and adhesion complexes are still forming.

Possible causes include:

Over-trypsinization or harsh pipetting during harvesting, leading to membrane damage.

Incomplete removal of trypsin or improper neutralization, resulting in continued enzymatic activity post-centrifugation.

Centrifugation at too high a speed or duration, causing physical damage or poor recovery.

Media change with temperature/pH imbalance (e.g., cold or CO₂-deprived media), stressing cells.

Low seeding density, which can impair reattachment and survival due to lack of cell–cell contact signals.

To prevent recurrence, use gentle handling, ensure complete inactivation of trypsin, centrifuge at low speed (e.g., 200–300 × g, 5 min), pre-warm all reagents, and allow cells to reattach in undisturbed conditions for at least 24 hours.

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