Theoretical and practical approaches ofHepatocyte 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

Continuous culturePrimary culture

Finite culture Indefinite cultureSingle cell type roughly thirty times of division, enhanced by growth factors.number of cell cycles corresponding to organism life span

It is nearly the same as finite but the cells here can divide indefinitely by transformation into tumor cells, they called cell line.

Normal cells cultured without any change in their division rate

Cell line

Normal Transformed Stem cell

Taken from a tumor tissue and culture as a single cell type

Normal cells underwent a genetic change to be tumor cells

They are Master Cells that generate other differentiated cell types

Comparison between normal and transformed cell lines

Normal TransformedPloidy Diploid Heteroploid

Transformation normal transformed

Tumorigenicity non tumorigenic

Anchorage yes no

Density limitation yes no

of growth

Mode of growth monolayer monolayer or suspension

Serum requirement high low

Growth rate slow rapid

Yield low (6 cells /ml) 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 linesCell line Cell type and origin

3T3 Fibroblast (mouse)

BHK 21* Fibroblast (Syrian hamster)

MDCK Epithelial cell (dog)

HeLa * 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 cellsThere 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 ofthe 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 marrowstem 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 extra-embryonic 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 KingdomAnimalia, 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 replicativesenescence.

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 activatethese 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 whenhTERT is exogenously expressed the cells are able to maintaintelomere 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 lines1- ATCC: American type culture collection

2- ECACC: European collection of cell culture