I'm not exactly sure what your 3 pictures are. Is the first one of the cells that didn't adhere? Is the third one your cells before you trypsinized? When you count your cells, you should always use trypan blue so that you can see viability before you replate.

If your cells looked like they do in picture 3 and all you wanted to do was replace the media, then just replace the media. No need to trypsinize and centrifuge.

Some Practical Tips for Culturing and Passaging Caco-2 Cells

1. Cell Morphology and Attachment Based on the provided images, the current batch of Caco-2 cells appears to be in good condition. It’s important to note that Caco-2 cells generally attach more slowly compared to many other common cell lines, especially after thawing or passaging. Therefore, it’s better to avoid frequent microscopic observations during the early attachment phase. Minimizing unnecessary disturbances can help reduce the risk of detachment and floating cells.

2. Serum Concentration For Caco-2 culture, the recommended serum concentration is typically around 20%, which supports both proper attachment and healthy growth. If the serum concentration is too low, the cells may have difficulty attaching and grow more slowly. On the other hand, excessive serum may interfere with the differentiation process. If you notice poor cell condition, one of the first things to check is the serum concentration and quality.

3. Floating Cells – Causes and Troubleshooting If you observe an increasing number of floating cells over time, especially large cell sheets detaching from the surface, this is a warning sign that needs attention. Several factors could contribute to this issue:

① Culture medium composition, particularly the batch and quality of the serum. Caco-2 cells are quite sensitive to serum quality.

② Culture conditions, such as CO₂ concentration, humidity, and temperature in the incubator — all of these need to remain stable.

4. The Tricky Part – Trypsinization and Passaging Trypsinization is a critical step for Caco-2, and it’s also one of the most challenging parts for this cell line. Caco-2 cells form very tight intercellular junctions and adhere strongly to the culture surface, making them difficult to detach. The longer they are cultured, the tighter these junctions become, and the harder it gets to digest them. Generally, the recommended digestion time is 5-10 minutes, but this is not a fixed rule — you need to adjust it based on cell density and culture duration. Complete dissociation into single cells is almost impossible for Caco-2; the goal is to obtain small cell clusters rather than single cells. If, after digestion, some cells have already detached while others still cling tightly to the surface, you can collect the detached cells first, centrifuge them, and then add fresh trypsin to the remaining adherent cells. Continue digestion in the incubator, checking the cells every minute to avoid over-digestion.

5. Environmental Details – Small but Important Tips A few minor but helpful tips: Caco-2 cells are quite sensitive to external disturbances. For example, placing culture flasks near the incubator door or opening the incubator too frequently can negatively impact their condition. Whenever possible, place your Caco-2 flasks further inside the incubator, in a more stable spot, to reduce environmental fluctuations.

Hi! My experience with Caco-2 is that there is always a part of the population that is not adherent, regardless of the confluency.