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Cell Culture
AO 04/2015
What is Cell Culture?
• Cell culture refers to the removal of cells from an animal or plant and
their subsequent growth in a favorable artificial environment.
• The cells may be removed from the tissue directly and disaggregated
by enzymatic or mechanical means before cultivation, or they may be
derived from a cell line or cell strain that has already been
established.
Primary Cultures
Epithelial cells
in culture,
stained for
keratin (red)
and DNA
(green)
• Primary cultures are derived directly from excised, normal animal
tissue and cultures either as an explant culture or following
dissociation into a single cell suspension by enzyme digestion.
• Such cultures are initially heterogeneous but later become dominated
by fibroblasts.
• The preparation of primary cultures is labour intensive and they can
be maintained in vitro only for a limited period of time.
• During their relatively limited lifespan primary cells usually retain
many of the differentiated characteristics of the cell in vivo.
Continuous Cultures
Cultured HeLa cells have been stained with
Hoechst turning their nuclei blue, and are
one of the earliest human cell lines
descended from Henrietta Lacks, who died
of cervical cancer from which these cells
originated.
• Continuous cultures are comprised of a single cell type that can be serially propagated in
culture either for a limited number of cell divisions (approximately thirty) or otherwise
indefinitely.
• Cell lines of a finite life are usually diploid and maintain some degree of differentiation.
• The fact that such cell lines senesce after approximately thirty cycles of division means it
is essential to establish a system of Master and Working banks in order to maintain such
lines for long periods.
• Continuous cell lines that can be propagated indefinitely generally have this ability
because they have been transformed into tumour cells.
• Tumour cell lines are often derived from actual clinical tumours, but transformation may
also be induced using viral oncogenes or by chemical treatments.
• Transformed cell lines present the advantage of almost limitless availability, but the
disadvantage of having retained very little of the original in vivo characteristics.
Culture Morphology
• In terms of growth mode cell cultures take one of
two forms, growing either in suspension (as single
cells or small free floating clumps) or as a monolayer
that is attached to the tissue culture flask.
• The form taken by a cell line reflects the tissue from
which it was derived e.g. cell lines derived from
blood (leukaemia, lymphoma) tend to grow in
suspension whereas cells derived from solid tissue
(lungs, kidney) tend to grow as monolayers.
Media and growth requirements
Medium requirements
Physiological parameters
• A. Bulk ions – Na, K, Ca, Mg, Cl, P, Bicarbonate or
CO2
• B. Trace elements – iron, zinc, selenium
• C. sugars – glucose is the most common
• D. amino acids – 13 essential
• E. vitamins – B, etc.
• F. choline, inositol
• G. serum – contains a large number of growth
promoting activities such as buffering toxic
nutrients by binding them, neutralizes trypsin and
other proteases, has undefined effects on the
interaction between cells and substrate, and
contains peptide hormones or hormone-like
growth factors that promote healthy growth.
• H. antibiotics – although not required for cell
growth, antibiotics are often used to control the
growth of bacterial and fungal contaminants.
• A. temperature – 37 °C.
• B. pH – 7.2-7.5 and osmolality of medium must be
maintained
• C. humidity is required
• D. gas phase – bicarbonate conc. and CO2 tension
in equilibrium
• E. visible light – can have an adverse effect on
cells; light induced production of toxic compounds
can occur in some media; cells should be cultured
in the dark and exposed to room light as little as
possible;
Cell line frequently used
Representative cell morphology of tumorassociated macrophages, TAM, fibroblast and lung
tumor cell. B. Immunofluorescent was used to
distinguish macrophage, fibroblast and lung tumor
cell with antibodies targeting CD68 (red), nuclei
stained with DAPI (blue).
Cell line classification
• Cell lines can be classified as
endothelial such as BAE-1,
epithelial such as HeLa,
neuronal such as SH-SY5Y, or
fibroblasts such as MRC-5 and
their morphology reflects the
area within the tissue of origin.
HeLa (http://www.cbsnews.com/news/the-immortalhenrietta-lacks/)
• Henrietta Lacks is best known as the source of cells that form
the HeLa line, used extensively in medical research since the
1950s.
• Henrietta Lacks was born on August 1, 1920, in Roanoke,
Virginia. Lacks died of cervical cancer on October 4, 1951, at
age 31.
• Cells taken from her body without her knowledge were used
to form the HeLa cell line, which has been used extensively in
medical research since that time.
• Lacks's case has sparked legal and ethical debates over the
rights of an individual to his or her genetic material and tissue.
HeLa
• The cells from Lacks's tumor made their way to the laboratory of
researcher Dr. George Otto Gey. Gey noticed an unusual quality in
the cells. Unlike most cells, which survived only a few days, Lacks's
cells were far more durable. Gey isolated and multiplied a specific
cell, creating a cell line. He dubbed the resulting sample HeLa,
derived from the name Henrietta Lacks.
The HeLa strain revolutionized medical research. Jonas Salk used the HeLa strain develop the
polio vaccine, sparking mass interest in the cells. Scientists cloned the cells in 1955, as demand
grew. Since that time, over ten thousand patents involving HeLa cells have been registered.
Researchers have used the cells to study disease and to test human sensitivity to new products
and substances.
Applications of cell culture
• Cell culture is one of the major tools used in cellular and molecular
biology
• providing excellent model systems for studying the normal physiology and
biochemistry of cells (e.g., metabolic studies, aging), the effects of drugs and
toxic compounds on the cells and mutagenesis and carcinogenesis.
• It is also used in drug screening and development and large scale
manufacturing of biological compounds (e.g., vaccines, therapeutic proteins).
• The major advantage of using cell culture for any of these applications
is the consistency and reproducibility of results that can be obtained
from using a batch of clonal cells.
• Model System:
• Cell culture are used as model system to study basic cell biology and biochemistry, to study the
interaction between cell and disease causing agents like bacteria, virus, to study the effect of drugs,
to study the process of aging and also it is used to study triggers for ageing.
• Cancer Research
• The basic difference between normal cell and cancer cell can be studied using animal cell culture
technique, as both cells can be cultured in laboratory. Normal cells can be converted into cancer cells
by using radiation, chemicals and viruses. Thus, the mechanism and cause of cancer can be studied.
Cell culture can be used to determine the effective drugs for selectively destroy only cancer cells.
• Virology
• Animal cell cultures are used to replicate the viruses instead of animals for the production of vaccine.
Cell culture can also be used to detect and isolate viruses, and also to study growth and development
cycle of viruses. It is also used to study the mode of infection.
• Toxicity Testing:
• Animal cell culture is used to study the effects of new drugs, cosmetics and chemicals on survival and
growth of a number of types of cells. Especially liver and kidney cells. Cultured animal cells are also
used to determine the maximum permissible dosage of new drugs.
• Vaccine Production:
• Cultured animal cells are used in the production of viruses and these viruses are used to produce
vaccines. For example vaccines for deadly diseases like polio, rabies, chicken pox, measles and
hepatitis B are produced using animal cell culture.
• Drug Screening and Development:
• Animal cell cultures are used to study the cytotoxicity of new drug. This is also used to find out the
effective and safe dosage of new drugs. Now these tests are being conducted in 384 and 1536 well
plates. Cell-based assay plays an important role in pharmaceutical industry.
•
• Genetically Engineered Protein:
• Animal cell cultures are used to produce commercially important genetically engineered proteins such as
monoclonal antibodies, insulin, hormones, and much more.
• Replacement Tissue or Organ:
• Animal cell culture can be used as replacement tissue or organs. For example artificial skin can be produced
using this technique to treat patients with burns and ulcers. However research is going on artificial organ
culture such as liver, kidney and pancreas. Organ culture techniques and research are being conducted on
both embryonic and adult stem cell culture. These cells have the capacity to differentiate into many
different types of cells and organs. It is believed that by learning to control the development and
differentiation of these cells may be used to treat variety of medical conditions.
• Genetic Counseling:
• Fetal cell culture extracted from pregnant women can be used to study or examine the abnormalities of
chromosomes, genes using karyotyping, and these findings can be used in early detection of fetal disorders.
• Genetic Engineering:
• Cultured animal cells can be used to introduce new genetic material like DNA or RNA into the cell.
These can be used to study the expression of new genes and its effect on the health of the cell. Insect
cells are used to produce commercially important proteins by infecting them with genetically altered
baculoviruses.
• Gene Therapy:
• Cultured animal cells can be genetically altered and can be used in gene therapy technique. First cells
are removed from the patient lacking a functional gene or missing a functional gene. These genes are
replaced by functional genes and altered cells are culture and grown in laboratory condition. Then
these altered cells are introduced into the patient. Another method is by using viral vector, functional
gene is inserted into the genome of viral vector and then they are allowed to infect the patient, in the
hope that the missing gene will be expressed with the help of the viral vector.