Establishing and maintaining cancer cell lines involves several steps and techniques. Here are the general methods used in the establishment and maintenance of cancer cell lines:

1. Tissue Collection: The process begins with the collection of tumor tissue from a patient or experimental animal. The tissue is typically obtained through surgical resection, biopsy, or in the case of animal models, by implanting tumor cells or tissues.
2. Primary Culture: The collected tumor tissue is enzymatically or mechanically dissociated into single cells or small cell clusters. These cells are then placed in a suitable culture medium that provides essential nutrients and growth factors. The primary culture allows the tumor cells to attach to the culture vessel and initiate growth.
3. Cell Proliferation: The tumor cells in the primary culture undergo proliferation, forming a heterogeneous population of cells. This initial culture period allows the cells to adapt to the laboratory environment and stabilize their growth characteristics.
4. Subculturing: As the cells reach confluency, they are detached from the culture vessel using enzymatic or mechanical methods and transferred to new culture vessels. This process, known as subculturing or passaging, ensures the continuous growth and expansion of the cell population.
5. Immortalization: To establish a long-term and immortalized cell line, the primary culture undergoes a process of immortalization. This is often achieved by introducing genetic alterations or viral elements that bypass the normal cell cycle controls and allow the cells to divide indefinitely. Examples of immortalization methods include the introduction of viral oncogenes or the expression of telomerase to prevent telomere shortening.
6. Characterization and Authentication: Proper characterization and authentication of the established cell line are crucial to ensure reliability and reproducibility of research results. This involves verifying the cell line’s identity and confirming its similarity to the original tumor. Authentication methods include short tandem repeat (STR) profiling and comparison to a reference database.
7. Cryopreservation: To maintain long-term storage and ensure the availability of the cell line, cryopreservation techniques are employed. The cells are frozen in cryoprotective solutions at ultra-low temperatures, typically in liquid nitrogen, which preserves their viability for an extended period. Cryopreserved cell lines can be thawed and resuscitated when needed for experiments.
8. Routine Culture and Quality Control: The established cancer cell lines are routinely cultured and maintained in appropriate culture media supplemented with necessary nutrients and growth factors. Regular monitoring of cell viability, growth rate, and contamination (e.g., bacterial or fungal) is essential. Cell lines should be periodically tested for mycoplasma contamination, and measures should be taken to prevent cross-contamination with other cell lines.

Overall, the establishment and maintenance of cancer cell lines require expertise in cell culture techniques, sterile laboratory practices, and genetic manipulation methods. These processes enable the continuous growth and availability of cancer cell lines for various research applications, including studying cancer biology, drug discovery, and personalized medicine.