Cell culture Flashcards
(11 cards)
Primary Cells
Primary cells are directly isolated from tissues via enzymatic or mechanical disaggregation and are cultured under conditions that allow initial expansion until confluence is reached. These cells retain many of the physiological, morphological, and biochemical properties of the tissue they were derived from. However, they are difficult to culture, often exhibit poor survival in vitro, and are sensitive to genetic manipulation. Most significantly, primary cells have a finite lifespan, undergoing a limited number of divisions (~20–30 population doublings) before entering senescence—a stable, irreversible cell cycle arrest.
Immortalised Cells
In contrast, immortalised cells can divide indefinitely (continuous cell line). They are typically derived from tumour tissues or are genetically modified to bypass senescence. Immortalised cells are easy to grow, cost-effective, and provide homogeneous populations that enhance reproducibility in experiments. However, their genetic instability, including aneuploidy and accumulated mutations, may reduce their resemblance to in vivo counterparts, potentially limiting translational value.
Immortalised Cells Example
An example of a widely used immortalised line is HeLa, derived from cervical cancer, which continues to divide decades after initial isolation. These cell lines are indispensable for high-throughput assays, drug screening, and large-scale production of biological products, but should be chosen carefully depending on the biological question.
Strategies for Immortalising Primary Cells
Immortalisation can occur spontaneously, particularly in tumour-derived cells, or it can be induced by manipulating telomere maintenance or disrupting tumour suppressor pathways. The two most commonly used methods involve human telomerase reverse transcriptase (hTERT) expression and viral oncogene-mediated inactivation of p53/Rb.
- hTERT Expression (Telomere Maintenance Strategy)
Telomeres are repetitive DNA-protein complexes (TTAGGG repeats bound by the shelterin complex) located at the ends of chromosomes. In somatic cells, each division results in telomere shortening, and once a critical limit is reached, DNA damage responses activate senescence or apoptosis.
The enzyme telomerase, particularly its catalytic subunit hTERT, maintains telomere length and is active in germline, stem, and cancer cells, but not most somatic cells. By introducing cDNA encoding hTERT, often via lentiviral transduction, into primary cells, researchers can extend their lifespan and promote immortalisation while retaining much of the original cell phenotype.
This method is relatively gentle and minimally disruptive to cellular function, making it ideal for applications requiring cells close to their in vivo state. However, success rates can be low, and additional genetic changes may still be required for full immortalisation.
- Viral Oncogene Inactivation of Tumour Suppressors
Another common approach involves disrupting tumour suppressor pathways, particularly p53 and Rb, which act as critical regulators of the cell cycle and apoptosis. In normal cells, p53 responds to DNA damage by halting the cell cycle or triggering apoptosis, while Rb controls the G1–S phase transition via repression of E2F transcription factors.
Viral oncogenes can inactivate these checkpoints, driving uncontrolled cell division. Notable examples include:
SV40 large T-antigen: inactivates both p53 and Rb.
HPV E6 and E7: E6 induces p53 degradation, and E7 binds and inactivates Rb.
Adenovirus E1A/E1B and Epstein–Barr virus proteins also act similarly.
This method is highly efficient and often achieves immortalisation rapidly, but it introduces foreign viral genes and may significantly alter cell function. It is frequently used in combination with hTERT to enhance success and stability.
Conclusion
Primary and immortalised cells each offer unique benefits and limitations in cell culture. While primary cells better model in vivo physiology, their limited lifespan restricts their use. Immortalised cells overcome this barrier but may not faithfully reflect the biology of their origin. Advances in molecular biology now allow researchers to immortalise cells through hTERT-mediated telomere maintenance or disruption of tumour suppressor pathways via viral oncogenes. Each approach must be carefully chosen based on the intended application and desired fidelity to the native cell type.
