Cell culture refers to growing cells outside of their natural environment in an artificial setting. Some key developments in cell culture include Wilhelm Roux demonstrating maintenance of living cells outside the body in saline buffer in 1885, and Ross Granville Harrison developing the first techniques of cell culture in vitro using frog embryonic tissue in 1907. In the 1920s, composition of salt solutions was formulated for cell cultures. The first cell line, called the "L cell line", was established by Earle in 1948 using cells from mouse tissue. Hayflick and Moorhead defined the finite lifespan of normal human cells in 1961. Cell culture remains an important tool in biomedical research today.

2. Maham Adnan
University of Central Punjan
Lahore, Pakistan

3. “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
May be derived from a cell line or cell strain that has already been
established.
Cell cultures have marked a major change in research during the last
decades due to the versatility of studies and field trials to which they
apply.

5. Cell theory is the historic scientific theory.
1. All living organisms are composed of one or more
cells.
2. The cell is the basic unit of structure and
organization in organisms.
3. Cells arise from pre-existing cells.
 3 scientists:
1. Theodor Schwann
2. Matthias Jakob Schleiden.
3. Rudolf Virchow

6. Wilhelm Roux (185–1924) demonstrated
that it is possible to maintain living cells
outside the body in saline buffer for a few
days.
Leo Loeb (1869–1959) evaluated a
technique called “tissue culture within
the body.”
Loeb was able to culture cells from inside
and outside body tissues.
Leo Loeb Wilhelm Roux

7. The American embryologist Ross Granville Harrison
(1870–1959) developed the first techniques of cell culture
in vitro.
Small pieces of living frog embryonic tissue were isolated
and grew outside the body.
Harrison's experimentations made the cell life “visible.”
Harrison introduced aseptic techniques in working with
cell cultures.
Ross Granville
Harrison

8. The hanging drop technique is a well-established method for examining living,
unstained, very small organisms.
The traditional procedure employs a glass slide with a circular concavity in the
center.
Into which a drop of fluid, containing the 'microorganisms', hangs from a coverslip.

9. By 1910, Montrose Burrows adapts the hanging drop method for working
with warm blood tissues.
Chicken plasma clot was used instead lymph.
Alexis Carrel established cell cultures of embryonic and adult tissues of many
species.
Applied other culture media including:
1. The diluted plasma with varying concentrations of salt solution
2. The use of serum.
Developed the first cell line called Immortal Cells.
The term “tissue culture” was defined for the first time in 1911.
“a plasmatic medium inoculated with small fragments of living tissues.”

10. The development of first practical cell culture flasks (in 1923), which were
called “D flasks”.
This culture flask (also called a D‐3.5 flask) had a diameter of 3.5 cm and was
made of PYREX glass.
New cell culture flasks allowed to culture cells in a larger medium volume
and made culture maintenance much easier.

11. Introduction of the aseptic techniques and Rous and Jones tissue
trypsinization technique.
Use of trypsin solution results in obtaining single cell suspension and
cells detachment for subculture.
The 3% trypsin solution was used for plasma digestion and did not
damage most cells.
When 5% trypsin solution was tested, obtained cells were dead.

13. The first cell line—the “L” cell line—was established by Earle in 1948.
Derived from subcutaneous mouse tissue.
Displayed quite different morphology from the origin of tissue.
In 50s and 60s,diploid cell lines were developed:
1. HeLa Cell line
2. MRC‐5 (Lung tissue of baby)
3. WI‐38 from human tissue
4. Vero (Verde—French for green and RenO—French for kidney) from simian tissue

14. HeLa Cell Line MRC‐5 Cell Line WI‐38 Cell Line

15. In 1951, Dr. Jones diagnosed Henrietta Lacks with cervical
cancer.
He send a sample to Dr. Gay.
Cultivated the cells and discovered that the cell line was
found to be remarkably durable
Presented cell division every twenty hours.
This cell line called HeLa (Henrietta Lacks) gave him the
best means to develop the poliovirus
Allowing the development of the Salk vaccine.
HeLa cells were quickly reproduced in almost any media.
One of the most precious resources for studies in cancer

17. The establishment of cell lines gives possibilities to determine differences
between cell lines culture and the primary cell cultures.

18. In the 1920s, composition of salt solutions was formulated for cell cultures.
Pannett and Compton (1924), Gay (1936), Earle (1943) or Hanks salts (1948).
Establishing formulas of salt solutions - first step to define cell cultures
requirements.
The scientists indentified the most needed components for cellular
metabolism.
Between 1932 and 1962, about 60 chemically defined media were worked
out.
Morgan, Morton and Parker developed media199, and Earle and his
coworkers worked out protein free media for L cell culture.

19. First effect of antibiotics on cell cultured in vitro was established in
the 1940s.
Herrell and coworkers found that the different preparation of
penicillin exhibited toxic action on mitosis.
Due to some impurities in penicillin preparation.
Keilova - influence of streptomycin directly on the explants of heart,
aorta and frontal bone of the chick embryo.
Firstly, incubator was used by Robert Koch in his microbiological
studies in the second half of the nineteenth century.

20. In 1961, Leonard Hayflick and Paul Moorhead defined
the finite life span of normal human cells.
Started research on the possible viral etiology of
human cancer.
Normal human embryonic cells were exposed to
cancer‐cell extracts.
Hayflick expected that normal cells would change
and display cancer‐like properties, but normal cells
did not grow any longer.

21. A few years later (in 1961), when working with Paul Moorhead,
he performed a series of experiments that validated Carrel's
theory.
Hayflick divided the time of cell culture into three phases.
On the basis of these experiments, Hayflick argued that normal
cells have a finite capacity to replicate.
As opposed to cancer cells (e.g., HeLa cell line) that are
immortal and display indefinite growth.

22. In cell cultures, the transformation may occur spontaneously.
Immortal cell populations were observed in many laboratories
from the early 1940s to the early 1960s.
Immortal cells arise spontaneously from normal cells, and
murine cell cultures are especially prone to that process
The first hybrid mammalian cells were obtained via viral fusion
in human and mouse cells in 1965 by Harris and Watkins.

23. In their work, they demonstrated that fusion of cells of
different species was possible.
Using a new technique of UV inactivation, Harris and
Watkins obtained heterokaryons from human HeLa cells and
Ehrlich ascites tumor cells from mice.

24. A series of multiple achievements was triggered in the
following decades.
In 1975, the Nobel Prize winners for Medicine in 1984,
Georges Kohler and Cesar Milstein, accomplished the first
monoclonal antibodies.
In 1969, Augusti and Sato set tumor lines of mouse nerve
cells (neuroblastoma) and isolate clones electrically excitable
with nerve prolongs.

25. By 1973, Graham and van-der-Eb introduced DNA into
mammalian cells in culture (Graham and Van Der Er.,
1973)
Formed the basis for the development of techniques for
incorporating genes into the cellular genome.
In 1992 American Type Culture Association, a bankcell was
formed.

26. At the end of the 90’s, one of the latest
scientific successes in the mammalian cell
culture that played a leading and decisive role
happened.
The cloning of a mammal by Wilmut,
Schnieke and colleagues.
Dolly the sheep was the great biological
landmark of the late twentieth century.
 Again animal cell cultures were transformed
into an essential tool for the progress of
science as a solution to many of the problems
of human health.

27. In terms of technological development, from the 50's, marketing tools of cell
culture started, turning this technique easy and reproductible, to become
one of the most powerful tools widely used in research and development
of biotechnology applied to pharmaceuticals.
Stem cells have the unique ability to self-renew or to differentiate into
various cell types in response to appropriate signals.
Accordingly, human stem cells are of special interest in medical research.
Embryonic stem cells have the ability to differentiate into more cell types
than adult stem cells.
The nature of stem cells necessitates the use of special stem cell culture
media and reagents.