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Caco-2 Cell Growth Essentials: Traits and Troubleshooting Tips

Source: PricellaPublished: 2024-03-11

Caco-2 (human colon adenocarcinoma cells) were isolated from a 72-year-old male with rectal carcinoma in situ. The Caco-2 cell spontaneously differentiates into intestinal epithelial-like cells under standard culture conditions and is a commonly used model for colon cancer cells. This article introduces the growth characteristics of Caco-2 cells and common troubleshooting tips.

Growth Characteristics

1. Island-like Growth

The boundary of Caco-2 cell clusters is smooth, with individual cell clones in the field of view resembling small islands on the sea.

2. Abundant Vacuoles

Caco-2 cell clusters often contain large vacuoles, which are inherent characteristics of the cells and are considered a normal phenomenon.

3. Difficult Digestibility

Caco-2 cells are tightly connected to each other, making it difficult to dissociate them. Typically, after adding trypsin, the periphery of cell clones detaches first while the central portion remains attached to the substrate. Subsequently, the entire clone detaches, but the cells do not disperse from each other. Digestion takes about 5-10 minutes, and these cells are challenging to disperse into individual cells. Digestion can be terminated when cells can be dispersed into small cell clusters.

4. Slow Adhesion

Caco-2 cells adhere slowly. Under standard culture conditions(Cat.No.:CM-0050), adhesion is typically completed within approximately 24 to 72 hours after seeding. It is recommended to refresh the medium on the third day after seeding.

5. Slow Growth

Caco-2 cells have a long passage cycle, primarily due to their slow adhesion and growth. Under a 1:4 split ratio, passaging is typically performed approximately once a week.

Common Issues

1. What to Do If Cells Do Not Adhere?

If Caco-2 cells fail to adhere, one approach is to adjust the fetal bovine serum (FBS) concentration in the culture medium. Typically, the FBS concentration in the Caco-2 cell culture medium is 20%. If adhesion is compromised due to a decrease in serum concentration, supplement the medium with additional FBS to restore it to 20%. Continue culturing for 1-2 days, and the cells should adhere. Additionally, check if the culture medium is alkaline (appears purple-red). Alkaline pH in the medium may hinder cell adhesion.

2. Can DMEM be Used Instead of MEM as the Culture Medium?

Yes, it can. DMEM (Dulbecco's Modified Eagle Medium) is formulated based on MEM. Caco-2 cells can grow normally under the conditions of DMEM supplemented with 20% FBS and 1% P/S.

3. What Happens If MEM Does Not Contain NEAA?

When using MEM without NEAA (non-essential amino acids) in the culture medium, the growth rate of Caco-2 cells may decrease, and there may be an increase in floating cells.

4. What to Do If Cells Are Not Digesting Properly?

If digestion takes 5-10 minutes and only a portion of the cells detach while the rest remain tightly adhered, you can transfer the detached cells to a centrifuge tube. Then, add fresh trypsin to the original culture vessel and return it to the incubator to continue digesting the remaining cells. Check every 1 minute until all cells detach.

5. What to Do If There Are Many Floating Cells?

A small number of bright cells floating or adhering to cell clones is normal and will eventually integrate into the clone and grow normally. Therefore, avoiding frequent cell observation can reduce the production of floating cells.

However, if the floating cells become increasingly severe during the culture process, or even form large clusters, it is essential to check for abnormalities in the culture medium composition and culture environment.


1. Caco-2 cells have slow adhesion, especially during the initial recovery phase. It typically takes 2-3 days for them to adhere, and vacuole formation may occur.

2. During passaging, ensure proper digestion of Caco-2 cells to facilitate adhesion. Otherwise, adhesion may be difficult.

3. Caco-2 cells tend to grow in clusters. The higher the cell density, the more prevalent surface vacuoles and dark spots will be observed.

4. When Caco-2 cells reach approximately 80% confluency, they form dense cell layers, which may not be visually apparent.

5. Overgrowth of Caco-2 cells severely impacts cell health, making them fragile and prone to cell death after passaging.

6. The longer Caco-2 cells are cultured, the longer the digestion time required during passaging.

Basic Information:

Cell Name

Caco-2 (Human Colorectal Adenocarcinoma Cells)


CaCo-2; CACO-2; Caco 2; 

CACO 2; CACO2; CaCo2; CaCO2;

 Caco2; Caco-2/ATCC


Male; 72 years old; colon, colorectal adenocarcinoma

Growth Characteristics

Adherent cells

Cell Morphology


Growth Medium

Caco-2 Cells Complete Medium (Cat.No.:CM-0050)

Recommended Split Ratio


Recommended Medium Change Frequency

Every 48-72 hours

Freezing Medium

General Freezing Medium (Cat.No.:PB180436)

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