The Cell Cycle & Cell Culture
The cell cycle is the sequence of events by which DNA replicates, the cell prepares to divide, divides, and prepares for the next division — formally, the period between one cytokinesis and the next. In actively growing cells, mitosis occupies only a small part of the cycle; most of it is interphase. Progression happens in response to signals, some from inside the cell and some from its neighbours.
INTERPHASE
Interphase consists of three phases:
- G₁ (first gap) - recovery from the previous division; the cell grows, doubles its organelles and accumulates the raw materials for DNA synthesis.
- S (synthesis) - DNA replication: each chromosome enters with one chromatid and leaves with two identical sister chromatids.
- G₂ (second gap) - the cell synthesises the proteins needed for division, including spindle components.
Not every cell keeps cycling. From G₁ a cell can step out into G₀, a resting phase. Quiescent cells are dormant and can re-enter G₁ later; senescent cells are ageing and never will. Differentiated cells often enter G₀ permanently — neurons are the standard example of cells arrested in G₀ that cannot divide.
CELL CYCLE — interphase (G₁, S, G₂) followed by the mitotic phase, guarded by three checkpoints, with a G₀ exit for resting cells (only quiescent cells return).
CHECKPOINTS
There are three major checkpoints along the cell cycle, at which intracellular and extracellular conditions must be met before the process continues. If the requirements are not met, the cycle halts — and if damage cannot be repaired, the cell is removed entirely.
| Checkpoint | When | What must pass |
|---|---|---|
| 1. G₁ | End of G₁, before S phase (DNA replication) | Intracellular conditions: DNA checked and mutations corrected, organelles doubled, cell size sufficient, nutrients, ATP and nucleotides stockpiled. Extracellular conditions: temperature, growth factors and hormones favourable |
| 2. G₂ | End of G₂, before mitosis (M phase) | DNA replication checked for errors and repairs made — if repair is impossible, the cell undergoes apoptosis; MPF levels must have risen |
| 3. M | Metaphase → anaphase | Every sister chromatid attached to spindle fibres via its centromere and correctly aligned along the metaphase plate; then cyclin breaks down, MPF levels fall, and anaphase (chromosome separation) begins |
The G₂ gatekeeper, mitosis promoting factor (MPF), forms when the protein cyclin binds the enzyme cyclin-dependent kinase (Cdk). Sufficient MPF pushes the cell into mitosis; once M phase begins, cyclin is degraded, MPF disappears, and the exit into anaphase is triggered.
FACTORS REGULATING THE CELL CYCLE
The rate of the cell cycle is affected by factors both internal (intrinsic) and external (extrinsic) to the cell:
| Internal factors (inside the cell) | External factors (from outside) |
|---|---|
| MPF formation from cyclin + Cdk | Anchorage dependence — cells must attach to the extracellular matrix or a surface to divide |
| Regulatory proteins encoded by proto-oncogenes and tumour suppressor genes, which act on the cell's own DNA | Density dependence — no space, no division |
| Surface-area-to-volume ratio — an important physical determinant of readiness to divide | Growth factors and hormones |
| Nutrient reserves (such as glycogen) | Nutrient availability; temperature and pH, which affect the enzymes of division |
Growth factors are a subset of cytokines — signalling molecules secreted by neighbouring cells or by distant glands and tissues, which bind receptors on the target cell's surface and trigger a cascade of intracellular and nuclear events leading to division. Examples include erythropoietin (increases red blood cell synthesis); hormones such as oestrogen and testosterone also stimulate division in their target tissues.
Apoptosis is programmed cell death — the controlled alternative to division. The membrane bulges irregularly (blebbing), the cell shrinks, and its contents are repackaged into apoptotic bodies that neighbouring cells engulf — no spillage, no inflammation.
WHEN REGULATION FAILS: CANCER
Failure of cell-cycle regulation can have serious consequences for the organism, such as uncontrolled cellular proliferation — cancer. A mutagen is any agent that changes an organism's genetic material: physical (X-rays, UV light, radioactive decay), chemical (reactive oxygen species, arsenic) or biological (viruses, certain bacteria, transposons). Mutagens that lead to cancer are classified as carcinogens.
Most cancers involve mutations in two classes of gene:
- Proto-oncogenes - encode proteins that stimulate the cell cycle; when mutated or over-expressed they become cancer-causing oncogenes — a stuck accelerator.
- Tumour suppressor genes - encode proteins that repress cycle progression and promote apoptosis; losing them removes the brakes.
The resulting tumour may remain in place (benign) or invade neighbouring tissue (malignant). Metastasis is the spread of cancer from the primary tumour to a new site, forming a secondary tumour — made of the same cell type as the primary, which determines which treatment will work.
CELL CULTURE
Cell culture is the technique of growing cells — prokaryotic, animal or plant — under controlled laboratory conditions. A cell culture is a single layer of cells which, through further growth, may become a tissue culture (layers of the same cell type). Historically, humans have cultured cells to produce beer, wine, bread (yeast cultures), cheese and yoghurt (bacterial cultures). Modern techniques exploit the fast generation time of cultures grown under in vitro (artificial) conditions compared with in vivo (in the living organism) conditions. Cell cultures are now used across several industries:
- Horticultural - propagation of plant seedlings.
- Medical - diagnosing infections, testing the effectiveness of antibiotics, industrial-scale production of insulin for diabetes treatment, and skin cultures grafted onto patients with severe burns.
- Cosmetic - cultured cells increasingly replace live animals in testing cosmetic substances, for ethical reasons.
There are two kinds of culture:
| Primary culture | Continuous (immortalised) line | |
|---|---|---|
| Source | Enzymatically isolated from tissue (e.g. HUVEC) | Random mutation or deliberate modification (e.g. HeLa) |
| Lifespan | Finite — normal cells divide a limited number of times | Indefinite proliferation |
Culture conditions must recreate the environment of the body — constant and optimal for enzyme function (reflecting in vivo physiological conditions), and sterile to prevent contamination:
- Temperature (~20–45 °C depending on the cells) and pH (~6.0–7.5).
- Controlled osmotic pressure and adequate O₂/CO₂ exchange.
- Hormones and growth factors.
- A nutrient substrate — amino acids, carbohydrates, vitamins, minerals.
- Light, if the cells are photosynthesising plant cells.
CELL CULTURE — a tissue sample is dissected, enzymes break down the extracellular matrix while the solution is stirred, and the separated cells are placed on an inert agar medium with nutrients and growth factors.
Establishing a human cell culture follows a standard sequence:
- Tissue selection - identify and remove a section of the tissue of interest.
- Dissection - trim away fatty and damaged cells.
- Disaggregation - separate the cells mechanically or with protein-digesting (proteolytic) enzymes that break down the extracellular matrix.
- Incubation and growth - culture the cells, refreshing the medium to clear debris.
- Separation and purification - selective media, differential attachment, or immunomagnetic beads isolate the wanted cell type.
Applications of cell culture include:
- Virology - cultivating viruses to study their infectious cycles and to produce vaccines (polio, rabies, chicken pox, hepatitis B, measles).
- Genetic engineering - commercial-scale protein production.
- Gene therapy - cultured cells carrying a functional gene can replace cells carrying a non-functional one.
The most famous culture is the HeLa line — cultured continuously since 1951 from the cervical carcinoma of Henrietta Lacks, the first human cell line ever established, and still central to virus, cancer and genetics research.
Limitations of cell culture:
- Normal cells have a finite doubling potential — only immortalised lines escape it.
- Contamination — viruses, microorganisms, even cross-contamination with other cell types; rich media grow everything, so sterile technique is essential and antibiotics are routinely added.
- Cultured cells can change their morphology, function or gene expression — they are not a perfect substitute for cells in a living organism.
- Cost, and the loss of interactions with other cell types present in real tissue.