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The CEACAM family comprises both membrane-bound and secreted glycoproteins. The membrane-bound forms are anchored to the cell surface via either a glycosylphosphatidylinositol (GPI) moiety or a transmembrane domain. Members anchored by GPI primarily include CEACAM5, CEACAM6, CEACAM7, and CEACAM8. CEACAM5 is widely expressed on the surface of various cell types, including endothelial cells, epithelial cells, and immune cells. In normal tissues, CEACAM5 functions primarily as a cell adhesion molecule, mediating both homophilic and heterophilic adhesion. The protein encoded by CEACAM5 can regulate cell differentiation, apoptosis, and cell polarity, thereby promoting tumor progression; consequently, it serves as a clinical biomarker for gastrointestinal cancers. In malignant cells, CEACAM5 is predominantly overexpressed on the surface of epithelial tumor cells, including those associated with colorectal cancer, gastric cancer, pancreatic cancer, gallbladder cancer, lung adenocarcinoma, small-cell lung cancer, breast cancer, bladder cancer, and ovarian cancer.

Figure 1. Recombinant CEACAM5 HEK293 stably expressing CEACAM5.

Cell Resuscitation
1)Rapidly thaw the frozen cells in a 37 °C water bath for approximately 60 seconds. Once thawed (which may take slightly less or more than 60 seconds), immediately transfer the cell suspension from the cryovial into a 15 mL centrifuge tube containing 10 mL of pre-warmed  HEK293 Human CEACAM5 Cell complete culture medium.
2)Centrifuge cells at 1000 rpm for 5 min to remove medium, then resuspend cells in 5 mL of pre-warmed complete medium.
3)Transfer the cell suspension into a T25 culture flask and incubate at 37 °C with 5% CO₂.
4)After approximately 24–36 hours, replace the medium or passage the cells to remove non-adherent dead cells.

Subculturing procedure
1)When the cell density reaches the appropriate confluency for passaging, wash the cells with PBS, then add 1 mL trypsin to detach the cells. When more than 80% of the cells detach upon gently tapping the culture flask, add complete culture medium to terminate digestion. Gently pipette to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

2)Discard supernatant after centrifugation. Resuspend cells in fresh medium to a single-cell suspension and transfer to a new culture flask for continued growth.

Cell Freezing
After trypsinization and centrifugation of cells from each T75 flask or 10 cm culture dish, discard the supernatant. Add 2 mL of cryopreservation medium (90% FBS + 10% DMSO), gently resuspend thoroughly, and aliquot into two cryovials. Immediately place the cryovials into a controlled-rate freezing container (e.g., Nalgene 5100-0001), fill with isopropanol to the indicated level, and store at −80 °C. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.