When working with MDA-MB-175 VII cells, which are a human breast cancer cell line, it is crucial to ensure that the revival and maintenance conditions are optimal for the cells to regain their epithelial morphology. One of the first steps is to check the cell culture conditions. It is important to use a suitable growth medium, such as RPMI-1640 supplemented with 10% fetal bovine serum (FBS), L-glutamine, and antibiotics like penicillin-streptomycin. Additionally, the quality of the FBS can significantly impact cell growth and morphology. It is also essential to ensure that the culture medium is at the correct pH, typically around 7.2 to 7.4, and that the cells are incubated at 37°C in a humidified atmosphere with 5% CO2.

During the revival process, proper techniques must be employed. When reviving frozen MDA-MB-175 VII cells, they should be thawed rapidly in a water bath at 37°C. After thawing, transfer the cells to culture medium and centrifuge to remove any dimethyl sulfoxide (DMSO) used for cryopreservation. Gently resuspend the cells in fresh medium and allow them to settle in a culture vessel. It is important to monitor the cells after revival, as they should be passaged before reaching confluence. Allowing the cells to become over-confluent can lead to a loss of their epithelial morphology. When passaging, use a gentle approach to avoid damaging the cells, and ensure that you use an appropriate trypsin-EDTA solution for detaching adherent cells.

Maintaining the correct cell density is also vital for promoting optimal growth. MDA-MB-175 VII cells should typically be subcultured when they reach 70-80% confluence, and splitting the cells at a ratio of 1:3 or 1:4 can help prevent over-confluence. If a lack of epithelial morphology is observed, consider plating the cells at lower densities to encourage better cell-cell interactions. Additionally, supplementing the medium with growth factors, such as epidermal growth factor (EGF), may promote epithelial characteristics and enhance growth.

Regular monitoring and assessment of the culture are critical to ensure the health of the cells. Keep an eye out for any signs of contamination, pH changes, or nutrient depletion, as these factors can adversely affect cell health and morphology. Using microscopy can help assess the morphology of the cells; healthy MDA-MB-175 VII cells should exhibit a typical epithelial morphology with a cobblestone-like appearance.

Environmental factors must also be considered. Ensure that the incubator is functioning properly and that CO2 levels are stable, as fluctuations in temperature or CO2 can negatively impact cell growth and morphology. Lastly, if issues persist despite following these guidelines, it may be beneficial to verify the identity of the cell line through STR profiling to ensure that you are working with authentic MDA-MB-175 VII cells. By adhering to these protocols and best practices, you may improve the revival and maintenance of MDA-MB-175 VII cells and facilitate the restoration of their epithelial morphology.