After the isolation and cultivation of human esophageal cancer tissue derived fibroblasts, further identification and functional analysis of the cells are required to ensure that their biological characteristics meet the experimental requirements. The following are the key steps for subsequent operations:

1. Observation of cellular morphology

-Regularly observe cell morphology using an inverted microscope. Primary fibroblasts are usually spindle shaped or polygonal, grow adherent to the wall, and have well stretched cytoplasm. If circular floating cells or abnormal aggregation appear, it may indicate contamination or poor cell status, and the culture conditions need to be re evaluated.

2. Immunofluorescence identification

-Immunofluorescence staining was performed using specific markers such as Vimentin and α - SMA. Fibroblasts should highly express Vimentin, and the positivity rate of α - SMA can reflect their degree of activation. Pay attention to setting up isotype controls to exclude non-specific binding.

3. Functional verification experiment

-Proliferation ability testing: Use CCK-8 or EdU methods to evaluate cell proliferation activity and compare the differences between cancer adjacent and cancer derived fibroblasts.

-Collagen secretion analysis: The secretion level of type I/III collagen is detected by ELISA or Western Blot to determine its pro fibrotic ability.

-Co culture experiment: Co culture fibroblasts with esophageal cancer cell lines (such as KYSE-150) and observe their effects on cancer cell invasion and migration (such as Transwell experiment).

4. Freezing and Resuscitation

-Select logarithmic growth phase cells, divide them into cryovials using a 10% DMSO solution, and transfer them to liquid nitrogen after programmed cooling. Rapid water bath melting during recovery, centrifugation to remove frozen solution, resuspended in high concentration serum culture medium, and changed after 24 hours.

Precautions

-Strict aseptic operation to avoid mycoplasma contamination;

-The recommended number of passages for primary cells should not exceed 5 to prevent phenotype drift;

-The functional experiment needs to be repeated at least 3 times to ensure data reliability.