Note on:

1. Stem cells,
2. Cell growth,
3. Cell metabolism and Product formation,
4. Advantages and Disadvantages of Animal cell culture,
5. Applications of Cell Culture
6. and Comparison of cell culture with model organisms.

What are stem cells?

Stem cells are the body's raw materials — cells from which all other cells with specialized
functions are generated. It can be referred to as the body's master cells. Stem cells are do not yet
have a specific role and can become almost any cell that is required. Under the right conditions
in the body or a laboratory, stem cells divide to form more cells called daughter cells. These
daughter cells become either new stem cells or specialized cells (differentiation) with a more
specific function, such as blood cells, brain cells, heart muscle cells or bone cells (stem cells
mature into cells in bones, heart muscle, nerves, and other organs and tissue) and can then be
implanted into a person. No other cell in the body has the natural ability to generate new cell
types.
Signals that influence the stem cell specialization process can be divided into external, such as
physical contact between cells or chemical secretion by surrounding tissue, and internal, which
are signals controlled by genes in DNA.
Stem cells offer great promise for new medical treatments. Stem cells also act as internal repair
systems of the body. They have the potential to be grown to become new tissue (specific cells)
for use to replace cells affected by disease (regenerative medicine). People who might benefit
from stem cell therapies include those with spinal cord injuries, Type-1 diabetes, Parkinson's
disease, amyotrophic lateral sclerosis, Alzheimer's disease, heart disease, stroke, burns, cancer
and osteoarthritis.

Characteristics of Stem cells.

Stem cells are:
 derived from numerous sources

 They are unspecialized, so they cannot do specific functions in the body

 have different potency / differentiation capacities / Versatile: they can divide into more
stem cells or can become any type of cell in the body

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  capable of self-renewal: cells can divide, reproduce and renew themselves over a long
time.
 Durable: they are contained throughout life.
Sources of stem cells
Stem cells originate from two main sources: adult body tissues and embryos. There are other
sources of stem cells
1. Embryonic stem cells. These stem cells come from embryos that are 3 to 5 days old. At this
stage, an embryo is called a blastocyst (or ball of cells) and has about 150 cells. (Embryonic
stem cells are obtained from early-stage embryos — a group of cells that forms when eggs
are fertilized with sperm at an in vitro fertilization (IVF) clinic but never implanted in
women's uteruses and can be used only when the embryo is no longer needed). The stem
cells are donated with informed consent from donors. The stem cells can live and grow in
special solutions in test tubes or petri dishes in laboratories.
These are pluripotent stem cells, meaning they can divide into more stem cells or can become
any type of cell in the body. This versatility allows embryonic stem cells to be used to
regenerate or repair diseased tissue and organs.
2. Adult stem cells. Also called tissue-specific or somatic stem cells. These stem cells are
found in small numbers in most adult tissues. Stem cells have been found in tissues,
including the brain, bone marrow, blood and blood vessels, skeletal muscles, skin, the liver
and fat.

Compared with embryonic stem cells, adult stem cells have a more limited ability to give rise
to various cells of the body. In the past, scientists believed adult stem cells could only
differentiate based on their tissue of origin. However, some evidence now suggests that they
can differentiate to become other cell types, as well. For instance, bone marrow stem cells
may be able to create bone or heart muscle cells, not just only to blood cells.

There are many types of adult stem cells; they include the following:
 Mesenchymal stem cells are present in many tissues. In bone marrow, these cells
differentiate mainly into the bone, cartilage, and fat cells. As stem cells, they are an
exception because they act pluripotently and can specialize in the cells of any germ layer.
 Neural cells give rise to nerve cells and their supporting cells—oligodendrocytes and
astrocytes.
 Haematopoietic stem cells form all kinds of blood cells: red, white, and platelets.
 Skin stem cells form, for example, keratinocytes, which form a protective layer of skin.

3. Adult cells altered to have properties of embryonic stem cells (Induced pluripotent stem
cells (iPS): Regular adult cells had successfully been transformed into stem cells using

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 genetic reprogramming. By altering the genes in the adult cells, they have been
reprogrammed to act similarly to embryonic stem cells, thereby preventing immune system
rejection of the new stem cells. It is not yet know whether using altered adult cells will cause
adverse effects in humans. For example, researchers have been able to take regular
connective tissue cells and reprogram them to become functional heart cells. In studies,
animals with heart failure that were injected with new heart cells experienced improved heart
function and survival time.

4. Perinatal stem cells. Researchers have discovered stem cells in samples of amniotic fluid
drawn from pregnant women for testing or treatment — a procedure called amniocentesis, as
well as umbilical cord blood. These stem cells have the ability to change into specialized
cells.

Types of Stem cells (according to their POTENCY)

Stem cells are derived from numerous sources and have different potency capacities (potential to
differentiate into other types of cells). POTENCY of a stem cell describes this flexibility.
Some stem cells have more potential than others. Embryonic stem cells are the most potent, as
their job is to become every type of cell in the body.

•Unipotent stem cells form only one type of specialized cell type; can only produce cells of one
kind, which is their own type.
•Multipotent stem cells can form multiple types of closely related family cells (cells of its
lineage) and tissue types. Adult hematopoietic stem cells, for example, can become red and white
blood cells or platelets
•Pluripotent stem cells can form most or all cell types in the adult.

•Totipotent stem cells: can differentiate into all possible cell types. They
can form all adult cell types as well as the specialized tissues to support development of the embr
yo (e.g., the placenta). They have total potential to develop into any cell in the body. The first
few cells that appear as the zygote starts to divide are totipotent.

• Oligopotent: These can differentiate into a few different cell types

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Uses of Stem cells / Stem cells applications.

 Increase understanding of how diseases occur. By watching stem cells mature into cells in
bones, heart muscle, nerves, and other organs and tissue, researchers may better understand
how diseases and conditions develop.

 Tissue regeneration and therapies: Stem cells can generate healthy cells/ tissues to replace
cells affected by disease (in transplant and regenerative medicine) under the right
conditions. Stem cells can be guided into becoming specific cells (new tissue) that can be
used in people to regenerate and repair tissues that have been damaged or affected by disease.
As an example, doctors have already used stem cells from just beneath the skin’s surface to
make new skin tissue. They can then repair a severe burn or another injury by grafting this
tissue onto the damaged skin, and new skin will grow back. In cases where a patient does not
have a sufficient amount of undamaged skin for skin graft treatment, skin cells are isolated
from a small biopsy and expanded in a specialized laboratory. Millions of cells can be grown
in a relatively short time and transplanted onto the burn wound to speed up healing.
Doctors now routinely use adult hematopoietic stem cells to treat diseases, such as leukemia
and sickle cell anemia,

 Cell deficiency therapy: people with Type-1 diabetes could receive pancreatic cells to
replace the insulin-producing cells that their own immune systems have lost or destroyed.

 Development and Testing of new drugs for safety and effectiveness. Instead of testing
drugs on human volunteers, researchers can use some types of stem cells to test the new
drugs for safety and quality. For the testing of new drugs to be accurate, the cells must be
programmed to acquire properties of the type of cells targeted by the drug. For instance,
nerve cells could be generated to test a new drug for a nerve disease. Tests could show
whether the new drug had any effect on the cells and whether the cells were harmed.

 Stem cells are useful not only as potential therapies but also for research purposes and
and scientific discovery.

Limitations / complications / potential problems with using stem cells in humans.

 Adult stem cells
i. Versatility problem: Adult stem cells may not be able to be manipulated to produce all
cell types, which limits how adult stem cells can be used to treat diseases.
ii. Adult stem cells are also more likely to contain abnormalities (genetic defects)
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 - due to environmental hazards, such as toxins,
- or from errors acquired by the cells during replication.
 Embryonic stem cells
i. Uncertainty in differentiation into the specific cell types desired.
ii. Irregular growth
iii. Might also trigger an immune response in which the recipient's body attacks the stem
cells as foreign invaders,
iv. The stem cells might simply fail to function as expected, with unknown consequences.

Cell growth

Cell growth refers to the increase in cell size (mass accumulation). Unrestricted cell growth
causes cancer. Various growth conditions such as the temperature, nutrients, cell density, and
drug treatment can block the cell cycle / division at various stages. Instead of unrestricted
growth, the cell growth curve shows that the cell population increases through three phases:
incubation period → exponential hyperplasia → stagnation period. During exponential growth,
cells demonstrate great variation in required cell density and doubling times that are highly
dependent on cell type and growth conditions. Cell death occurs in each generation.

Stages of cell growth.

Cellular growth usually shows three sequential steps: incubation, exponential hyperplasia, and
stagnation/death phases.
Cells grow by cell division. The cycle a cell undergoes as it grows and divides is a 4-stage
process consisting of Gap 1 (G1), synthesis (S), Gap 2 (G2), and Mitosis (M) phases in sequence.
The G1, S, and G2 stages are known collectively as the interphase and they are the stages
between one mitosis and the next. During interphase, the cell grows and makes a copy of its
DNA. During the mitotic (M) phase, the cell separates its DNA into two sets and divides its
cytoplasm, forming two new cells. After completing the cycle it either starts the process again
from G1 or exits through G0. From G0, the cell can undergo terminal differentiation.

 The signature of the G1 phase is the synthesis of enzymes that are required for DNA
replication.

 During the S phase, DNA is replicated to produce two identical sets of chromosomes.

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  The G2 phase is mainly involved in the production of microtubules that are required
during the process of division, the mitotic phase. Increases in cell volume are observed
during the interphase (G1, S, G2 phase).

 The M phase consists of prophase, metaphase, anaphase, and telophase in sequence, and
the parent cell is divided into two daughter cells through nuclear division (karyokinesis),
cytoplasmic division (cytokinesis), and formation of a new cell membrane.

Causes of cell growth

The combined influence of growth factors, hormones, and nutrient availability provides the
external cues for cells to grow.

Problems with cell growth

1. The larger the cell, the more demands the cell places on its DNA.
2. The cell has trouble moving enough oxygen, nutrients and wastes across the cell membrane.
The advantage of cells being small is that materials can be distributed to all parts of the cell
quickly.

Major factors that limit cell growth.

1. The single factor that limits the size of most of the cell is the surface volume ratio. The size of
the cell gets limited by the nucleo-cytoplasmic ratio, which determines the range of control of
metabolic ratio and the ratio of surface to volume.
3. Cell growth is subject to changes in disease state
4. and /or environmental conditions.

What stops cell growth

Senescence — a permanently static state, in which cells stop growing and dividing — results
from serious stress and genetic damage. GATA4 (a buildup of the protein) helps turn on this
response in cells.
In healthy, dividing cells, GATA4 is quickly gobbled up and broken down. But this cleanup
process slows down in damaged cells, causing the protein to build up and initiate senescence.

Monitoring cell growth

Direct methods to determine the cell growth and/or viability in a bioprocess include microscopic
counting, electronic particle counting, image analysis, in situ biomass monitoring, and
dieletrophoretic cytometry. These methods work most simply when a fixed volume sample can
be taken from a suspension culture.

Measurement of cell growth rate

Many times investigators want to determine how quickly cells are dividing mitotically or
growing (sometimes referred to as a doubling rate). A simple method to measure the rate of

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growth is to count the number of cells in a small volume (aliquot) at several time points and plot
a growth curve.

Cell metabolism and Product formation

Cellular metabolism is the set of enzyme-mediated chemical reactions that occur within each
cell of living organisms in order to maintain life. These processes allow organisms to grow and
reproduce, maintain their structures and respond to environmental changes.
Purposes of metabolism
The three main purposes of metabolism are
 the conversion of food/fuel to energy to run cellular processes,
 the conversion of food/fuel to building blocks for proteins, lipids, nucleic acids, and some
carbohydrates,
 and the elimination of nitrogenous wastes
Classification of metabolism
Cellular metabolism has two distinct divisions: anabolism and catabolism. Anabolism is a
constructive metabolic process in which a cell uses energy to construct molecules such as
enzymes and nucleic acids and perform other essential life functions. Anabolism involves three
basic stages: firstly, the production of precursors such as amino acids, monosaccharides,
isoprenoids and nucleotides; secondly, their activation into reactive forms; and thirdly, the
assembly of these precursors into complex molecules.
Catabolism is the metabolic process by which the cell breaks down complex molecules. The
purpose of catabolic reactions is to provide the energy and components needed by anabolic
reactions. Catabolic reactions are normally exothermic and are further subdivided according to
their substrate into carbohydrate, fat, and protein catabolism.
Regulation and Control of cellular metabolism
This is by a sequence of enzymes. Enzymes are crucial to metabolism and allow the fine
regulation of metabolic pathways to maintain a constant set of conditions in response to changes
in the cell's environment, a process known as homeostasis.

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Several hormones of the endocrine system also help control the rate and direction of
metabolism. Thyroxine, a hormone made and released by the thyroid gland, plays a key role in
determining how fast or slow the chemical reactions of metabolism go in a person's body.
Another gland, the pancreas, secretes hormones that help determine whether the body's main
metabolic activity at any one time are anabolic or catabolic. For example, more anabolic activity
usually happens after you eat a meal. That's because eating increases the blood's level
of glucose — the body's most important fuel. The pancreas senses this increased glucose level
and releases the hormone insulin, which signals cells to increase their anabolic activities.

Products of cellular metabolism

The reactants and products of these chemical reactions are metabolites which include water,
carbon dioxide, and energy (ATP), urea (end product of protein metabolism in mammals)

Advantages or benefits of using animal cell culture:

1. Cell culture allows for the alteration of various physiological and physiobiological conditions
such as pH, temperature and Osmotic pressure.
2. Animal cell culture is a great way to study issues concerning cell metabolism and helps to
understand the cell biochemistry.
3. The results of animal cell culture are consistent regardless of the type of cell that is utilized.
4. This technique can also facilitate the identification of various cells on the basis in the
appearance of marker, such as molecules, or through the karyotyping.
5. Manages ethical issues on animal usage as this is a barrier to the animal experiments.
6. Animal cell cultures can be utilized to produce massive amounts of antibodies and proteins
that otherwise would require an investment of a substantial amount.
7. Production of cells in large scale for research uses and commercial purposes.

Disadvantages of Animal cell culture

 It is a highly specialized method that requires highly skilled personnel / experienced
personnel
 It is a costly procedure because it requires expensive equipment.
 Risk of contamination as the techniques must be performed using strict asepsis
techniques.
 Additionally, there is a difficulty in maintaining the cells viability in vitro conditions, as
animal cells may not survive when isolated and therefore are not capable of an independent
sustainable existence without providing a complex environment.
 Subcultures of the cell line could result in different properties when different from that of
the initial strain.
 The process produces a relatively low amount of cells which increases the cost of the
procedure.
 The cells produced by this technique lead to instability due to the occurrence of
aneuploidy chromosomal constitution.

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Use / Applications of Cell Culture
Animal cell culture has many potential applications:
 Providing excellent model systems for studying the normal physiology and biochemistry
of cells (e.g., metabolic studies, aging),
 It is also used in drug discovery and development (the effects of drugs and toxic
compounds on the cells).
 Large scale manufacturing of biological compounds (e.g., vaccines, therapeutic proteins).
 tissue engineering,
 and basic research.

Comparison of cell culture with model organisms.

Biology research relies on the use of model systems. Both animal models and cultured cells are
necessary, with advantages and disadvantages. For some research goals, only one type of model
system will be used.
Some issues of concern include:
 Cost and availability: The cost of using whole animals has risen considerably as the
Federal government continues to issue regulations for the care and maintenance of the
animals. The difficulty in obtaining animals has raised the cost of each cat or dog so high
that most laboratories cannot afford to use them. Therefore many researchers have turned
to isolated-organ or cell culture.
 Scaling: Cultured cell experiments are highly scalable; more experiments can be done
more rapidly than using animals. For example, potential cancer drugs are typically tested
on large panels of hundreds of different cell lines with known mutations. This would not
be feasible with animals, but animal models allow testing on intact physiological systems
of many cell types simultaneously. For example, mouse models of cancer or diabetes can
be used to study the effects of a drug, or other perturbation, on blood sugar, kidney
function, inflammation, and other effects, all within one animal.
 Using whole animals may take considerable time; it usually takes one whole day to do
one experiment. Organ or tissue culture experiments are usually done with multiple
samples at one time, and for a shorter duration than with the whole animal
 Visualizing: Cells grown in culture can be easily observed using standard microscopes,
and the use of modern fluorescent labels even allows imaging of membrane diffusion,
cell adhesion, signal transduction, and other cellular processes at the molecular level. By
contrast, mice and rats are bulky and opaque (i.e. large, complex and dense), making
cellular activities very difficult to observe in vivo. However, we now understand that
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 many cellular responses are strongly affected by intercellular signaling within tissues,
which limits the usefulness of cell culture data. Also, new techniques are now making the
imaging of cellular and molecular dynamics in live tissues increasingly feasible. In
particular, intermediate systems, such explant culture models, offer many of the benefits
of cell culture experiments, while preserving the complex in vivo cellular environment.
 Translating: It is obvious that rodents are more similar to humans than are cultured cells,
and because of this, animal models can be used to probe aspects of biology that are more
relevant to human health. For example, animal models of tumor metastasis or heart
disease do not have any cultured cell model equivalent. But, it is important to note that
human and rodent physiology is not identical, and even sophisticated animal models can
have shortcomings.
 In the whole animal, there are many mechanisms that act to regulate each physiological
mechanism, some stimulatory and some inhibitory. In cell culture it is possible to study
one of these at a time, but there is no way to study all of the possible interactions that can
occur in physiological circumstances.

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