Cell Cycle and Cell Culturing

Cells as the Basis of Life

Cell Cycle and Cell Culturing

SCIENCE UNDERSTANDINGS

Cell division may be regulated by internal and external factors.
The cell produces gene products that regulate the cell cycle.
● Describe the stages in the cell cycle (including checkpoints).
● Explain that hormones may regulate cell division.
Carcinogens upset the normal controls of cell division by causing mutations in key
regulatory genes.

Human beings culture cells for a variety of purposes.
● Describe techniques of cell culture, and discuss the applications and limitations of
contemporary examples.

• The cell cycle has three main parts—interphase, mitosis and cytokinesis
• As the cytoplasm divides during cytokinesis, this marks the beginning of the

2 new cells.

• The cell cycle is the period between one cytokinesis & the next. In actively

growing cells, mitosis occupies only a small part of the cycle.

• Each part of the cell cycle involves a number of sequential steps
• The progress of a cell from one step to the next occurs in response to

certain signals. These signals may come from within the cell itself or from
other cells in its environment

Cell Cycle

Most of the cell cycle is spent in interphase.
G1 Phase – pre-DNA synthesis:

• Recovery from previous division
• Cell doubles its organelles
• Accumulates raw materials for DNA synthesis

S Phase:

• DNA replication (synthesis)
• Chromosomes enter with 1 chromatid each
• Chromosomes leave with 2 identical chromatids each

G2 Phase:

• Between DNA replication and onset of mitosis
• Cell synthesizes proteins necessary for division

Interphase

G0 Phase- resting phase (quiescent)
● cell temporary or permanently leaves

the cell cycle

● It exits at G1

G0 phase

× Not all cells are continually replicating – some may enter into a non-dividing

G0 stage

× These cells may either be dormant (quiescent) or ageing and deteriorating

(senescent)

× Cells enter the G0 phase from the G1 phase; quiescent cells may re-enter

G1 at a later time (senescent cells do not)

× Normally, cells will only divide a finite time before reaching senescence

× Specialised cells will often permanently enter G0, as differentiation has

prevented their capacity for further division

× Neurons are examples of cells that have been arrested in a G0 state – these

cells cannot divide

Cell Cycle Checkpoints

CHECKPOINT

LOCATION IN THE
CELL CYCLE

DETAILS

G1

At the end of the
G1 phase, prior
to the S phase of
interphase

Ensures the cell is ready for cell division. DNA is checked and mutations are corrected. Cell
must have doubled the organelles and accumulated nutrients, ATP and DNA nucleotides.
Growth factors and hormones trigger the processes needed to enable the cell cycle to
move through G1. The cell remains at this checkpoint if any of these requirements for cell
division are not met.

G2

At the conclusion
of the G2 phase,
prior to the cell
entering mitosis

DNA replication has occurred and is checked for errors (mutations). Repairs are made, if not
able to be repaired it will go through programmed cell death (apoptosis).
For a cell to continue through G2, maturation promoting factor (MPF) must be synthesised.
This requires sufficient amounts of both cyclin-dependent kinase (Cdk) and the protein
cyclin. A complex between the kinase and cyclin produce MPF. When sufficient MPF is
present the cell continues into the M phase.

M

Towards the end
of metaphase,
prior to anaphase

Sister chromatids; are assessed for any issues prior to separation, are attached via the
centromere to the spindle fibres at each pole.
This triggers a breakdown of the cyclin protein, which causes a reduction in the
concentration of MPF present. This triggers the entrance into Anaphase.

The cell cycle is regulated by a number of factors determining if a cell is able to
begin or complete its cycle.

Cell Cycle

INTERNAL FACTORS

EXTERNAL FACTORS

Located within a cell

Act from outside of the cell

Formation of maturation promoting factor (MPF),
due to the presence of enzymes (cyclin-dependent
kinase) and protein (cyclin)

Can be for both chemical and physical factors that control the cell cycle.
Can trigger internal factors

Cyclin and other proteins that control the cell cycle
are encoded by proto-oncogenes and tumour
suppressor genes.

Physical: Anchorage dependence. Cells must be anchored to the extracellular
matrix or physical surface to divide.

Surface area to volume ratio (size of the cell).
Efficiency of obtaining nutrients and removal of
wastes. Large cells will divide more readily than
smaller cells.

Physical: Density Dependence. Cells require space to divide into. If there is no
space then the cells will not divide.

Chemical: Growth Factors and hormones. These chemicals regulate the cell cycle.

Nutrient availability: needed to ensure enough raw materials are available for the
cell to grow.

Growth Factors act as a signalling molecule between the cells.

●

Growth factor is a subset of cytokines. It can be secreted by
neighbouring cells as well as distant glands and tissues.

●

Growth factors bind their receptors on the target cells’ surface.

However, there are a few factors that could affect the growth of target cells
such as;

●

the type of receptors,

●

transduction of intracellular signal,

●

number of target cells,

●

the binding ability of growth factor,

●

growth factor concentration

External control factors- Growth Factors

Cell cycle checkpoints help guide the cell through the cycle and ensure that no mistakes or errors are
carried onto the next stage.

Maturation promoting factor (MPF) serves as a G2 checkpoint and helps regulate the passage from
the G2 to M phase.
● when both cyclin and cyclin dependent kinase (Cdk) are present at the end of the G2 stage, they

form a complex of MPF, which facilitates the transition to the M phase.

● Once the M phase has started, cyclin will be degraded and MPF will no longer be active.

Internal control factors

Apoptosis is programmed cell death (‘cell
suicide’)

Apoptosis is a controlled event regulated by
molecular signals which inhibit or promote
this process.

The plasma membrane undergoes irregular
bulging, or blebbing, and cell contents are
repackaged for safe removal.

The cell shrinks and converts fragments into
apoptotic bodies which are subsequently
engulfed by neighbouring cells.

Apoptosis

A mutagen is an agent that changes the genetic material of an organism (either
acts on the DNA or the replicative machinery)

Mutagens may be physical, chemical or biological in origin:
● Physical – Sources of radiation including X-rays (ionising), ultraviolet (UV) light and

radioactive decay

● Chemical – DNA interacting substances including reactive oxygen species (ROS) and

metals (e.g. arsenic)

● Biological – Viruses, certain bacteria and mobile genetic elements (transposons)

Mutagens that lead to the formation of cancer are further classified as
carcinogens

Carcinogens

An oncogene is a gene that has the potential to cause cancer

Most cancers are caused by mutations to 2 basic classes of
genes

1. Proto-oncogenes code for proteins that stimulate

the cell cycle and promote cell growth and proliferation

2. Tumour suppressor genes code for proteins that

repress cell cycle progression and promote apoptosis

When a proto-oncogene is mutated or subjected to increased
expression it becomes a cancer-causing oncogene

Oncogenes

Tumour cells may either remain in their original location (benign) or spread and invade
neighbouring tissue (malignant).

Metastasis is the spread of cancer from one location (primary tumour) to another,
forming a secondary tumour.

Secondary tumours are made up of the same type of cell as the primary tumour – this
affects the type of treatment required

Metastasis

Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown
under controlled conditions.

Cell Culturing

Primary Cultures (e.g. HUVEC, smooth muscle cells)

• Enzymatically isolated from tissue
• Finite lifespan

Continuous/Immortalised Cell lines (e.g. HeLa)

• Random mutation or deliberate modification
• Indefinite proliferation

Primary and Continuous Cultures

The conditions will be different for different types of cells, but in general, the culture will
require:
● controlled temperature. The best temperature will vary on the type of cells you are

growing, but it will probably be between 20-45 C.

● controlled pH. The best pH will likely be somewhere between 6.0 - 7.5.
● controlled osmotic pressure
● adequate exchange of oxygen and carbon dioxide
● hormones
● growth factors
● a substrate that will provide the cells with the nutrients they need. This includes

amino acids, carbohydrates, vitamins, and minerals.

● Light. Light is crucial if you are growing plant cells which must photosynthesize

Conditions for Cell Culturing

The conditions will be different for different types of cells, but in general, the culture will
require:
● controlled temperature. The best temperature will vary on the type of cells you are

growing, but it will probably be between 20-45 C.

● controlled pH. The best pH will likely be somewhere between 6.0 - 7.5.
● controlled osmotic pressure
● adequate exchange of oxygen and carbon dioxide
● hormones
● growth factors
● a substrate that will provide the cells with the nutrients they need. This includes

amino acids, carbohydrates, vitamins, and minerals.

● sterile environment
● light is crucial if you are growing plant cells which must photosynthesize

Conditions for Cell Culturing

carbon

Tissue of interest
●

Identify the tissue of interest- remove a section of the tissue

Dissection
●

Process the tissue, removing fatty and damaged cells

Disaggregation (separation)
●

Use a mechanical or enzymatic treatment to separate the cells

Incubation and growth
●

Incubate separated cells, change the growth medium to remove loose
debris or unattached cells

Separation and purification
●

Further purification of cells of interest achieved by selective media,
removal of cells at different levels of attachment, and with specific
immunomagnetic beads

Process of obtaining Human Cells for Cell Culture

The limitations of cell culture include the finite doubling potential of most
normal cells,
The possibilities for unexpected infection with viruses or microorganisms, or
even cross-contamination with other cell types.
Media used to propagate cells are rich in nutrients and, therefore, support
growth of a multitude of organisms. Accordingly, most culture methods require
sterile conditions.
Often antibiotics are used to inhibit growth of unwanted microbial
contaminants.
Another difficulty with some cultured cells is their tendency to change their
morphology, functions, or the range of genes they express.

Cell Culturing- limitations

Virology - Cultivation of virus for vaccine production, also used to study their infectious
cycle.

Genetic Engineering - Production of commercial proteins, large scale production of
viruses for use in vaccine production e.g. polio, rabies, chicken pox, hepatitis B &
measles

Gene therapy - Cells having a functional gene can replace cells which have a
non-functional gene

Cell Culturing- applications

HeLa cell, a cancerous cell belonging to a strain
continuously cultured since its isolation in 1951
from a patient suffering from cervical carcinoma.

The designation HeLa is derived from the name of
the patient, Henrietta Lacks.

HeLa cells were the first human cell line to be
established and have been widely used in
laboratory studies, especially in research on
viruses, cancer, and human genetics.

HeLa Cells