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Group 4 Project - Cell Culture - CellBiology
Group 4 Project - Cell Culture - CellBiology
Contents
1 Introduction
2 History
2.1 Ross Granville Harrison (1870 - 1959)
2.2 Alexis Carrel (1873 - 1944)
2.3 Charles Lindbergh (1902 - 1974)
3 Methodology
3.1 Harvesting and Isolating Cells
3.2 Growth Media For Cell Cultures
3.3 Important Cell Culture Techniques
4 Applications
4.1 Plant Tissue Culture
4.2 Importance of Plant Tissue Culture in Biotechnology
4.3 Application of Organ Culture
5 Limitations
5.1 Potential Cell Culture Problems
5.2 Limitations of Haploids
5.3 Limitations of Micropropagation
5.4 Limitations of Somatic Embryogenesis
6 Current Research & Advances
6.1 Current Success and Future Directions in Ovarian Tissue Culture
6.2 Gene Transcription in the Mammary Gland
6.3 Culturing Cells on a Lab Chip
6.4 The Immune Response
7 Glossary
8 Helpful Links
9 References
9.1 2010 Projects
Introduction
Cell Culture is the process where cells are derived from an organism, such as a plant or animal and is placed
within an artificially controlled environment to stimulate growth. The most common methods of cell culture are
tissue and organ culture. The availability of appropriate nutrients and conditions will allow cells that are removed
from various tissues and organs to continue to develop in vitro, where the cell acts as an independent unit. Cells
developed in vitro will continue to divide, increase in size and grow until affected by an external condition, such as
nutrient depletion. Cells within culture can either be genetically identical or may show genetic variation.[1] Cell
cultures consist of primary cultures, semi continuous cell cultures and continuous cell cultures.
The process of cell culture consists of the isolation of cells, the maintaining of cells within culture, the cross
contamination of a cell line, the manipulation of cultured cells, establishing human cell lines and the development of
a generation of hybridomas.[2] The technique of cell culture allows for numerous applications, such as the
investigation of the biochemistry of cells, the generation of artificial tissues and has made significant impact in the
research of virology through the testing of chemical compounds and drugs on specific cell types. Cell culture (/cellbiology/index.php/File:ZEpith
research has assisted in manufacturing antibodies, vaccines and cell-produced drugs.[3] Cells.jpg)
Epithelial Cells in Culture
History
Ross Granville Harrison (1870 - 1959)
Cell Culture arose in the 19th century when physiologist Sydney Ringer developed a salt solution that was able to sustain the beating of an animal
heart outside the body, however the principle of tissue culture was first evident in 1885 when Ross Granville Harrison and Wilhelm Roux established
the method known as tissue culture.
From 1907-1910 Harrison established the methodology of tissue culture, which became a common technique in the mid 1900s. Harrison utilized the
process of tissue cell culture to analyze nerve fibres in vitro. Harrison extracted neural tube and lymph fragments from frog embryos and observed the
development of the nerve fibres on a glass slide.[4]
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(/cellbiology/index.php/File:ZCell_Culture_Timeline.JPG)
Significant Landmarks in Cell Culture
was further refined to lead to the development of the world’s first heart and lung machines and contributed to the first open heart surgery in the 1950s.
This glass chamber consisted of PYREX® glass that eliminated contamination issues and was nontoxic. “The perfusion pumps I designed had to be
made of a special glass that would not crack under the heat of sterilization or dissolve to the extent ordinary glass would dissolve in perfusing fluids.
Such glass was sold under the name Pyrex“[6]
Throughout the 1940-1950s, John Franklin Enders, Thomas Huckle Weller & Fredrick Chapman Robbins conducted significant advances in cell culture
techniques, which assisted in virology research, where viruses were developed within cell cultures to manufacture vaccines. Bioreactors were created
in the 1950s to produce large quantities (holding approximately 20, 000 liters) of antibodies and cell-produced drugs, where cells performed their ideal
function within highly controlled and monitored environmental conditions. Today, cell culture is a significant principle for research. In 2006 the US
Congress divided $1 billion to various pharmaceutical companies to develop cell cultured influenza vaccines and there have been significant advances
through the establishment of various centers that specialize and support cell culture research.[7]
Methodology
Cell cultures are divided into three general classes which are Primary cell cultures, Semicontinous cell cultures (low-passage cell lines) and Continuous
cell cultures (immortalised cell cultures). The differences between these cell cultures arise from where the cells were taken from. Primary cell cultures
consist of cells derived directly from the host organism. The semicontinuous cell cultures are obtained from subcultures of a primary culture and usually
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consist of diploid fibroblasts that undergo a finite number of divisions. Continuous cell cultures are derived from transformed cells that are generally
epithelial in origin. They are classified this way because these cultures grow rapidly, are heteroploid (having a chromosome number that is not a simple
multiple of the haploid number) and can be subcultured indefinitely, which is unlike the other two cell culture classes.[8]
2. Tissue samples are treated with proteolytic enzymes (eg. trypsin and collagenase) to dissociate
either cell adhesion proteins or to degrade the matrix if it is digestable.
If an enzyme is used to dissociate the cells from the source then the resulting suspension would be
spread over a flat surface (eg. bottom of culture flask, or petri dish) in order to culture the cells.
There are many types of different growth media available and are either defined or undefined media. Defined media is growth media in which all the
quantities of the ingredients are known, whereas undefined media has some complex ingredients and all the ingredients involved are in unknown
amounts. Growth media can be varied in pH, glucose concentration, growth factors and the presence of different nutrients. The factors which can be
varied can also be used to help isolate and grow a particular type of cell. Apart from the growth media cells also need the right temperature and
appropriate gas mixture to be able to grow. The conditions must be ideal for the particular cell which is being cultured in order for the cell culture to be
successful.[18]
A cell incubator is used in order to keep the cells in a desirable state to allow them to proliferate. Most human cells are placed into cell culture
incubators with a temperature of 37°C, relative humidity of 95% and 5% carbon dioxide[19].
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Ability to
Allows easy visualisation of cells Harder to view cells
see cells
Used for cytology, harvesting products Used for bulk protein production, batch
Uses continuously and also research harvesting and also research
applications applications
Cells need to be cultured using very sterile techniques to avoid cross-contamination of the cell type of interest with other cell types. This is done by
using aseptic technique which is a process in which the equipment used to culture the cells are constantly sterilised and the process limits the chance
of contamination[23]. There is no guarantee however that this technique is perfect and thus it is recommended that the researchers always verify the
cells which they have cultured and make sure it is the cell type of interest. Once verification is complete and the cell of interest has been found it can be
subcultured to let that cell type to proliferate.
Applications
Cell culture has beneficial uses to help improve human health. Many different vaccines have been made with the
help of this technique which include measles, mumps, rubella, polio and hepatitis A. Therapeutic proteins have also
been made using cell cultures of animal cells as hosts. Some of these proteins include Erythropoietin (EPO), Tissue
plasminogen activator (TPA) and γ-interferon. The technique of cell culture has also found use assisting the
production of cell therapy products and gene therapy products.[24]
Plant tissue culture now has direct commercial applications as well as value in basic research into cell biology,
genetics and biochemistry. The techniques include culture of cells, anthers, ovules and embryos on experimental to
industrial scales, protoplast isolation and fusion, cell selection and meristem and bud culture. Applications include:
cross-pollinate distantly related species and then tissue culture the resulting embryo which would otherwise
normally die,for production of doubled monoploid plants from haploid cultures to achieve homozygous lines more (/cellbiology/index.php/File:Huma
rapidly in breeding programmes, usually by treatment with colchicine which causes doubling of the chromosome Human cell lines
number, certain techniques such as meristem tip culture can be used to produce clean plant material from virused
stock, such as potatoes and many species of soft fruit, micropropagation using meristem and shoot culture to
produce large numbers of identical individuals, to cross distantly related species by protoplast fusion and
regeneration of the novel hybrid, as a tissue for transformation, followed by either short-term testing of genetic
constructs or regeneration of transgenic plants. [25]
A plant breeder may use tissue culture to screen cells rather than plants for advantageous characters, e.g. herbicide resistance/tolerance. Large-scale
growth of plant cells in liquid culture inside bioreactors as a source of secondary products, like recombinant proteins used as biopharmaceuticals.
Micropropagation is widely used in forestry and in floriculture. Micropropagation can also be used to conserve rare or endangered plant
species.Somatic embryogenesis which is the development of embryos from vegetative cells rather than from union of male and female gametes.[26]
The two main applications of plant tissue culture in horticultural production are in propagation and sanitation. Tissue culture is being used effectively in
asexual clonal propagation of various horticultural crops. Orchids, carnations, roses, gerbera, chrysanthemums, potatoes, strawberries, apples and
coffee have all been successfully cloned in vitro and the list is increasing daily. Recovery of pathogen - free plants by meristem tip culture has become
a common practice in producing virus-free stock material of vegetatively propagated plants. Other areas where tissue culture can contribute to
horticulture include embryo culture and germplasm preservation and storage.[27]
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Biotechnologists are also trying to modify the genetics of cultured cells in three ways:
(i) mutagenesis and selection of cell lines in cell suspension culture, (ii) transplantation of foreign
genetic material in protoplasts by means of genetic engineering, and (iii) somatic hybridization by
the fusion of distantly related plant protoplasts just to widen the genetic diversity of hybrids.[29].
The basis of explant is also the patient itself or the prepuce of new born babies. The use of a synthetic polymer
PGA, permits the new born skin to grow without scars. This artificial skin is used to cover the wound until the
patient's skin is cultured and artificial skin is acquired for gratfing.
The keratinocytes manufacturing up the bulk of the epidermis is trypsioized. These cells are cultured in vessels,
the bottom of which is covered with irradiated 3T3 fibroblast cell line. Propagation of keratinocytes is inspired by
certain products from fibroblasts. Keratinocytes form colonies, which is again dissociated and cultured. The
process is repeated till a nonstop sheet of pure epithelium is made. This sheet is detached, cleaned and used
for grafting. Rejection of implant can be evaded by taking the explant from the patient itself. A 1.7 m2 artificial
skin can be acquired from a 3 cm2 skin in 34 weeks-a SOOO-foid increase. Artificial skin graft have been used
to fix effectively several skin defects, skin ulcers, etc. All essential components of skin will be regenerated in
about 5 years, after the grafting of the artificial skin.[31]
In the field of mutagenesis and selection of cell lines in vitro and the exploitation of totipotency, biotechnologists
are trying to improve plants e.g. by producing crop species that are more resistant to drought, disease, poor soil
conditions, chemical pesticides and herbicides.Another goal of biotechnology using the plant tissue culture
technique is to produce plants that would provide their own usable nitrogen. [32] (/cellbiology/index.php/File:Tissue_cu
Tissue culture growth medium
Limitations
Expertise is needed, so that behaviour of cells in culture can be interpreted and regulated.
Ten times more expensive for same quantity of animal tissue; therefore reasons for its use should be compelling.
Unstable aneuploid chromosome constitution.[33]
There are various ways to avoid these problems by manipulating the cell cultures. To replace nutrients depleted the growth media can be removed and
then replaced. To overcome the problem of senescence a subculture can be performed which involves transferring a small number of cells into a new
culture dish. Both animal cells and plant cultures can be cultured but the appropriate growth media and conditions must be met. A major difference
between plant and animal cell culture is the ability of many of the plant cells, with appropriate signalling and manipulation, to form any of the cell types
or tissues of a plant[35]. This feature is known as totipotency.
Limitations of Haploids
In many crops, the application of this technique is not yet possible since the technique for haploid production is not available.
In many other crops, its application is not feasible because large numbers of haploid plants are not easily obtained.
High cost of obtaining haploids and doubled haploids is still a major problem.
A nonrandom recovery of haploids may reduce the spectrum of variability recovered.
A sophisticated tissue culture laboratory and a dependable greenhouse are essential for success.
Specialized skill for carrying out the various operations is required.
Occurrence of grametoclonal variation may limit the usefulness of pollen embryos for genetic transformation/gene transfer.
High frequency of albinos are produced in anther cultures of monocots, especially cereals.[36]
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Limitations of Micropropagation
Expense
Species specificity
Production scheduling
Contamination
Variability
Acclimatization [37]
Experiments on ovarian tissue from non-human primate models and from agreeable fertile and infertile patients benefit from a multidisciplinary
approach. The new discipline of oncofertility needs professionalization, multidisciplinarity and mobilization of funding for basic and translational
research. [39]
These cultures have a limited lifespan and are not useful for studies at the cellular level. Epithelial cells can be isolated from mammary tissue,
maintained in culture and induced to distinguish with lactogenic hormones. The applications of such primary cultures has established the importance of
the cellular substratum in the differentiation process. A major disadvantage of this system, in addition to the short lifespan of the cells is the
considerable amount of starting material required.[40]
Glossary
Aneuploid – an abnormal number of chromosomes
Callus Culture - a tissue culture technique that requires growth regulators and extracts from an organ or tissue culture
Centrifugation - an instrument that rotates at high speed to separate substances of different concentrations
Continuous/Immortalised Cell Cultures – cells that are derived from transformed cells that are generally epithelial in origin
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Diploid – a cell with double the number of chromosomes
Epigenetic – the process where genetic information is modified by environmental influences, which is expressed within the behaviour of an organism
Glycosides – an organic compound derived from plant to produce a sugar and non-sugar compound
Growth Media – a liquid or gel designed to support the growth of microorganisms or cells
Haploid – a cell with a complete set (half the number of a diploid cell) of chromosomes
In Vitro - where a procedure occurs within a controlled external environment rather than in the living organism
Micropropagation – a plant tissue culture technique, where offspring are cloned from the tissue of a single plant
Primary Cultures – cells that are derived directly from a host’s tissue or organ
Sanitation – the process and application of measures to ensure the prevention of disease and contamination
Semicontinuous Cell Cultures/Low-Passage Cell Lines – cells that are obtained from subcultures of the cells of primary culture
Somaclonal – the term used to describe genetic variation that arises within plants due to plant tissue culture
Subculture – a cell culture made by transferring cells from a previous culture to a fresh growth medium
Helpful Links
ATCC - The Global Bioresource Center that focuses in Cell Culture http://www.atcc.org/ (http://www.atcc.org/)
European Collection of Cell Cultures (ECACC) that preserves and distributes cell lines http://www.hpacultures.org.uk/collections/ecacc.jsp
(http://www.hpacultures.org.uk/collections/ecacc.jsp)
National Cell Culture Center (NCCC) that produces cell lines for fundamental research http://www.nccc.com/ (http://www.nccc.com/)
PubMed- a datatbase that provides research articles related to Cell Culture http://www.ncbi.nlm.nih.gov/sites/entrez
(http://www.ncbi.nlm.nih.gov/sites/entrez)
Tissue Engineering Replacing organs or tissues with lab-created counterparts; engineered kidneys, livers and hearts http://www.youtube.com/watch?
v=ofiLcTs7_Ys&feature=related (http://www.youtube.com/watch?v=ofiLcTs7_Ys&feature=related)
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2010 Projects
Fluorescent-PCR (/cellbiology/index.php/Group_1_Project_-_Fluorescent-PCR) | RNA Interference (/cellbiology/index.php/Group_2_Project_-
_RNA_Interference) | Immunohistochemistry (/cellbiology/index.php/Group_3_Project-_Immunohistochemistry) | Cell Culture | Electron Microsopy
(/cellbiology/index.php/Group_5_Project_-_Electron_Microsopy) | Confocal Microscopy (/cellbiology/index.php/Group_6_Project_-
_Confocal_Microscopy) | Monoclonal Antibodies (/cellbiology/index.php/Group_7_Project_-_Monoclonal_Antibodies) | Microarray
(/cellbiology/index.php/Group_8_Project_-_Microarray) | Fluorescent Proteins (/cellbiology/index.php/Group_9_Project_-_Fluorescent_Proteins) |
Somatic Cell Nuclear Transfer (/cellbiology/index.php/Group_10_Project_-_Somatic_Cell_Nuclear_Transfer)
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