Download Normal Transformed Stem cell

Theoretical and practical approaches of
Hepatocyte primary culture
Workshop
14-16 February 2006
Lecture (3)
Cell lines: Sources importance and applications
Coarse organizer
Dr. Abo bakr Mohamed Eltayeb
Types of tissue culture
Primary culture
Normal cells
cultured
without any
change in their
division rate
Continuous culture
Finite culture
Single cell type roughly
thirty times of division,
enhanced by growth
factors.number of cell
cycles corresponding to
organism life span
Indefinite culture
It is nearly the same
as finite but the
cells here can
divide indefinitely
by transformation
into tumor cells,
they called cell line.
Cell line
Normal
Taken from a tumor
tissue and culture as a
single cell type
Transformed
Normal cells
underwent a genetic
change to be tumor
cells
Stem cell
They are
Master Cells
that generate
other
differentiated
cell types
Comparison between normal and transformed cell lines
Normal
Transformed
Ploidy
Diploid
Heteroploid
Transformation
normal
transformed
Tumorigenicity
non
tumorigenic
Anchorage
yes
no
Density limitation
yes
no
Mode of growth
monolayer
monolayer or suspension
Serum requirement
high
low
Growth rate
slow
rapid
Yield
low (6 cells /ml)
of growth
high (> 106 cells/ml)
Although all cell lines are similar, they are often not
identical. The genetic uniformity of a cell line can be
improved by cell cloning, in which a single cell is isolated
and allowed to proliferate to form a large colony. So a
clone is any such collection of cells that derived from a
single ancestor cell. Most cell lines used nowadays are
clones. Some cell lines are used from relatively long time as
U937 which isolated from a human with leukemia, this cell
line is used from almost forty years ago. One of the most
important uses of cell cloning has been the isolation of
mutant cell lines with defects in specific genes. Studying
such cell lines often reveals a good deal about the function
of that gene and its protein in normal cells.
One of the most famous story about normal cell lines is
HeLa cell line, HeLa cells have been isolated from a 31
years old woman from Baltimore USA, here name is
Henrieta Lacks, a doctor isolated some cells from her
neck and send them to the lab. To check if she has cervical
cancer. The result indicated that the cells are really
malignant. The woman died 8 months latter, but here cells
still used in the laboratories since 1951 till now.
HeLa cell line in culture
The most commonly used cell lines
Cell line
3T3
BHK 21*
MDCK
HeLa *
Cell type and origin
Fibroblast (mouse)
Fibroblast (Syrian hamster)
Epithelial cell (dog)
Epithelial cell (Human)
PtK1
Epithelial cell (rat Kangaroo)
L6
myoblast (rat)
PC 12
Chromaffin cell (rat)
SP2 *
Plasma cell (mouse)
U937 *
Monocyte (Human)
*Cells are capable to grow in suspension, the other cell lines require
a solid culture substratum in order to multiply.
Stem cells
There is a third type of cell lines called stem cell line, Stem cells are
nothing but “Master Cells” that generate other differentiated cell
types. Each tissue within the body contains a unique type of stem
cells that renew and replace that tissue (e.g. nerve, brain, cartilage,
blood) when needed due to damage or wear and tear. Stem cells of
the blood (hematopoietic stem cells) generate all other blood cells in
the human body, including red blood cells, platelets, and white blood
cells. Sources of hematopoietic stem cells include umbilical cord
blood, bone marrow, peripheral blood and embryos. In other
simpler words, stem cells are the body's "master" cells because they
give rise to all other tissues, organs, and systems in the body. The
stem cells' ability to differentiate, or change, into other types of cells
in the body, is a new discovery that holds tremendous promise for
treating and curing some of the most common diseases such as heart
disease, cancers, Alzheimer's and many others.
Types of Stem Cells
Generally, stem cells are categorized according to their source, as
either adult or embryonic. The type of stem cell most often discussed
in the news is an embryonic stem cell. During fertilization, a sperm
cell unites with an egg cell, and begins to reproduce by dividing into
different cells. These cells begin to arrange themselves into an outer
ring of cells that enclose an inner cell mass called a blastocyst.
Researchers have collected these inner cells and discovered that they
can be made to develop into many types of specialized cells in the
body.
These cells from the inner cell mass contain embryonic stem cells,
which is an accurate term because they do come from the first stages
of an embryo. But once these cells are removed from the inner cell
mass, they are not able to develop into an infant. Adult stem cells are
undifferentiated cells found among differentiated cells of a specific
tissue and are mostly multipotent cells. They are already being used
in treatments for over one hundred diseases and conditions.
Until recently it was thought that each of these cells could produce
just one particular type of cell—this is called differentiation.
However in the past few years, evidence has been gathered of stem
cells that can transform into several different forms. Bone marrow
stem cells are known to be able to transform into liver, nerve, muscle
and kidney cells.
Embryonic stem cell research is a less-developed field and is
considered by many researchers to have greater potential as the
basis of treatments. Embryonic stem cells are cultured cells obtained
from the inner mass cells of a blastocyst. Research with embryonic
stem cells is controversial because it requires destruction of embryos
same like abortion, which to many people are human beings,
meaning that destroying an embryo for any reason is morally
unacceptable.
Recently there are four types of stem cells
Totipoten
Which are capable of forming every type of body
cell. Each totipotent cell could replicate and differentiate and
become a human being. All cells within the early embryo are
totipotent up until the 16-cell stage or so. A single totipotent stem
cell can grow into an entire organism and even produce extraembryonic tissues.
Pluripotent stem cells cannot grow into a whole organism, but
they are able to differentiate into cells derived from any of the three
germ layers. In cell biology, a pluripotent cell is one able to
differentiate into many cell types. In the members of Kingdom
Animalia, pluripotent stem cells which can develop into any of the
three major tissue types: endoderm (interior gut lining), mesoderm
(muscle, bone, blood), and ectoderm (epidermal tissues and nervous
system). Pluripotent stem cells can eventually specialize in any
bodily tissue, but they cannot themselves develop into a human
being.
Multipotent (also called unipotent) stem cells can only become
some types of cells: e.g. blood cells, or bone cells. Although limited in
number, but multipotent stem cells can give rise to several other cell
types. An example of multipotent cells is hematopoietic cells—blood
stem cells that can develop into several types of blood cells.
cloned stem cell, or therapeutic the stem cell line, where a non
stem cell from blood or other tissue is allowed to fuse with an egg
ghost (ovum without nucleus) after fusion the result egg has only one
nucleus, it is the one of the blood cell and after stimulation of the
new ovum to start its cleavage, one cell of the early developed
embryo (usually before 32 blastomere stage) is taken and allowed to
grow in appropriate medium. This type of stem cells is a promising
and newly developed research field for solving many diseases.
Make your own cell line
Cell immortalization
Why don't cells live forever?
After a series of population doublings (the number of which varies
by species, cell type, and culture conditions) primary cells enter a
state where they no longer divide. This state is called replicative
senescence.
Replicative senescence is marked by distinct changes in cell
morphology, gene expression, and metabolism and can be induced
by extrinsic factors, intrinsic factors, or both. Extrinsically,
irradiation, oxidative stress, bring about senescence by triggering
the activation of various tumor suppressor proteins, including p53,
Rb, and P16/INK4A.
Intrinsically, the telomeric ends of chromosomes shorten with each
mitotic cycle and eventually the short or uncapped ends activate
.these same tumor suppressor proteins, inducing senescence.
How can cells be made immortal in culture?
Several methods exist for immortalizing mammalian cells in culture,
the most important are: viral transformation ,Viral genes,
including Epstein-Barr virus (EBV), Simian virus 40 (SV40),
adenovirus E1A and E1B, can induce immortalization by a process
known as. viral genes achieve immortalization by inactivating the
tumor suppressor genes.
Telomerase transcriptase inhibitors
TERT transfection ,telomerase reverse transcriptase enzymes
particularly those cells most affected by telomere length (e.g.,
human). This protein is inactive in most somatic cells, but when
hTERT is exogenously expressed the cells are able to maintain
telomere lengths sufficient to avoid replicative senescence. Analysis
of several telomerase-immortalized cell lines has verified that the
cells maintain a stable genotype and retain critical phenotypic
markers.
Importance and applications of cell lines
1- Mutant cell production
2- Advace in cancer ubderstanding
3- Progress in cell biology
4- Cord blood technology, leukemia , transplant rejection, ID
diseases bone healing etc.
5-SCNT technology
6-Drug discovery and metabolism. ETC…..
Two main purchasing sources of cell lines
1- ATCC: American type culture collection
2- ECACC: European collection of cell culture