2.11.

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Cell and Tissue Culture

History of Cell Culture
Contents:

End of
Live Under the
Main Milestones Spontaneous The Cell Theory
Microscope Generation

Harrison’s Hanging Cell Culture

Drop Technique Techniques and Hayflick Immortality of
Establishing
and Dr. Carrel’s Phenomenon HeLa Cell Line
Immortal Cells Principles in Cell
Culture Maintaning
Ece ÖZYAĞCI
Selay TORNACI
Immortalization Phenomenon of
Ebru TOKSOY ÖNER of Cells and First Induced Future of Cell
Monoclonal Pluripotent Stem Cultures
Antibodies Cells
Marmara University, Department of Bioengineering

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Cell and Tissue Culture Cell and Tissue Culture

History of Cell Culture – Main Milestones History of Cell Culture – Main Milestones

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History of Cell Culture - Live Under the History of Cell Culture - Live Under the
Microscope Microscope
Netherlands
• Hans Janssen and his son (around In the first publication (1653), Petrus Borellus
In the 16th and 17th centuries 1590) invented a compound described the use of microscope in medicine.
microscope
Italy Athanasius Kircher (1601-1680) showed the
• Galileo Galilei (1564-1642) living creatures in decaying tissues in 1658.
constructed «occhialino» (around
1610). In 1667, Swammerdam (1637-1680) and
• In 1625, the term «microscope» Malpighi (1628-1694) characterized red blood
used by Giovanni Faber. Figure 3. «Vase»
cells. microscope used by
Figure 1. Hans Malpighi
Janssen and his son’s
compound
Campini used the microscope firstly for the
microscope Figure 2. Galileo clinical examination.
Galilei

Kriss, T. C., & Kriss, V. M. (1998). History of the operating microscope: from magnifying glass to
microneurosurgery. Neurosurgery, 42(4), 899-907. Bradbury, S. (2014). The evolution of the microscope. Elsevier .

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History of Cell Culture - Live Under the History of Cell Culture - Live Under the
Microscope Microscope
Robert Hooke (1635-1702) Anton Von Leeuwenhoek (1632-1732) – Father of microbiology

• Constructed lenses that can magnify 270x and examined many samples
• First to use histological staining (muscle tissue with saffron)

Hooke published the

Micrographia in 1665.

Today’s term «cell»

comes directly from the
Hooke’s Micrographia.

Figure 4. A. Drawing of the microscope of

Figure 6. A. One of the microscopes of Leeuwenhoek.
Robert Hooke. B. Drawing of a cork in Hooke’s Figure 5. Anton Von B. Protozoa image from one of the microscopes of
Micrographia. Leeuwenhoek
Leeuwenhoek.
Kriss, T. C., & Kriss, V. M. (1998). History of the operating microscope: from magnifying glass to Kriss, T. C., & Kriss, V. M. (1998). History of the operating microscope: from magnifying glass to
microneurosurgery. Neurosurgery, 42(4), 899-907. microneurosurgery. Neurosurgery, 42(4), 899-907.

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History of Cell Culture - Live Under the History of Cell Culture - End of Spontaneous
Microscope Generation
In the 20th century, Aristotalian doctrine of spontaneous generation: generation of insects from
• First phase contrast microscope mud, flesh, and other inorganic or organic matter.
(1941): Fritz Zernike
• Differential interference contrast
technique was created by Georges
Nomarski.
• In 1957, confocal microscopy
technique was evolved by Marvin Figure 7. Figure 8. Phase contrast
Fritz Zernike image of primary cultured
Minsky. neurons
• Super resolution fluorescence
microscopy was developed by Stefan
Hell, in 1990s.
Figure 10. Illustration of Francesco Redi’s experimantal
setup tested in 1668, «Experiments on the Generation of
Frits Zernike – Nobel Lecture. NobelPrize.org. Nobel Prize Outreach AB 2023. Mon. 2 Oct 2023.
https://www.nobelprize.org/prizes/physics/1953/zernike/lecture/> Insects».
Retrieved from
Kuriyama, K., & Ohkuma, S. (1990). Cerebral cortical neurons in primary culture and application to neuropharmacological
https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_(OpenStax)/03%3A_The_Cell/3.01%3A_Spontaneous_
studies. In Methods in neurosciences (Vol. 2, pp. 103-116). Academic Press.
Generation

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History of Cell Culture - End of Spontaneous History of Cell Culture - End of Spontaneous
Generation Generation
Louis Pasteur (1822-1895) laid the spontaneous generation doctrine.
He showed that the microorganisms are on the dust and makes the broth cloudy when
they are exposed to dust.
In the 18th century, Italian
Lazzaro Spallanzani showed that
a living organism was derived
from another living organism by
repeating the experiments of
English John Turberville
Needham.

Figure 11. Lazzaro Figure 13. The swan necked

Figure 12. Louis Pasteur observing the
Spallanzani (1729-1799) flask designed by Pasteur.
culture broth in a swan necked flask.
Photo courtesy of Institut Pasteur.
Bordenave, G. (2003). Louis Pasteur (1822–1895). Microbes and infection, 5(6), 553-560.
Burget, G. E. (1924). Lazzaro Spallanzani (1729-1799). Annals of Medical History, 6(2), 177 . Mahadevan, S. (2007). The legend of Louis Pasteur. Resonance, 12, 15-22 .

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History of Cell Culture - End of Spontenous History of Cell Culture - The Cell Theory
Generation
Felice Fontana (1730-1805): first description of nuclei in
epithelial cells published in 1781.
After Pasteur’s experiments, Joseph Robert Brown (1773-1858): The term nucleus («little nut» in
Lister (1827-1912) developed antiseptic latin) was introduced in 1831.
surgical procedures.
Matthias Schleiden (1804-1881) and Theodor Schwann
He used carbolic acid as an (1810-1882) expressed the «cell theory». Their «free cell
antibacterial agent. formation» theory relying on spontaneous generation was
rejected by Albert Kölliker, Rudolf Virchow, and Robert Remark.

Schwann defined the three essential elements (nucleus,

Figure 14. Joseph
membrane or wall, and a fluid content) of a cell and decided that
Lister (1827-1912) cells arise inside and near other cells by differentiation of
cytoblastema.
Michaleas, S. N., Laios, K., Charalabopoulos, A., Samonis, G., & Karamanou, M. (2022). Joseph Lister (1827-1912): A Pioneer
of Antiseptic Surgery. Cureus, 14(12), e32777. https://doi.org/10.7759/cureus.32777

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History of Cell Culture - The Cell Theory History of Cell Culture - Harrison’s Hanging Drop
Technique and Dr. Carrel’s Immortal Cells
Virchow presented that the cells are
formed by splitting of preexisting cells. In In the late 19th century,
1858, he stated that a plant arises only
from plant and an animal arises only from Wilhelm Roux showed that cells
animal. can be cultured outside the body
(in vitro) for a few days in saline
In cellularpathologie, he mentioned that buffer.
all the diseases are the consequence of Figure 17. Leo Loeb
Leo Loeb performed the tissue
changes in normal cells. culture within the body by (1869-1959)

Figure 15. Rudolph grafting tissues and and fluids in

Figure 16. Wilhelm
Virchow (1821-1902) Roux (1850-1924)
living animals.

Sander, K. (2012). Landmarks in developmental biology 1883–1924: historical essays from Roux’s Archives. Springer Science & Business
Media.
Schultz M. (2008). Rudolf Virchow. Emerging Infectious Diseases, 14(9), 1480–1481. https://doi.org/10.3201/eid1409.086672 Retrieved from https://www.aacr.org/governance/leo-loeb/

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Cell and Tissue Culture Cell and Tissue Culture

History of Cell Culture - Harrison’s Hanging History of Cell Culture - Harrison’s Hanging
Drop Technique Drop Technique
In the beginnig of 20th
Table 1. Pros and cons of Harrison’s hanging drop technique
century,
Ross Granville Harrison Hanging Drop
(1870-1959) used the first in
vitro animal cell culture Pros Cons
technique called «hanging
drop». • Simply assambled and do not • Airtight sealed and burdensome
He adapted the technique, require a special equipment. for longtime cultivation.
invented by Robert Koch (in • Can be used for microscopic • Fluidic control is out-of-hand.
1880s), used in imaging. • Media-to-cell ratio is low.
microbiology with bacteria. • Handy closed system. • Tissue bulks are prone to
Due to rapid • Suitable for histological necrosis.
contaminations, he needed Figure 18. Hanging drop technique a. scheme b. photograph preparations.
c. growing nerve fiber of a frog, and a red blood cell marks a • Contamination is restricted
to develope the aseptic
fixed point, in the hanging drop culture, drawn by Harrison. with individual cultures
techniques.

Millet, Larry & Gillette, Martha. (2012). Over a Century of Neuron Culture: From the Hanging Drop to Microfluidic Devices. Millet, Larry & Gillette, Martha. (2012). Over a Century of Neuron Culture: From the Hanging Drop to Microfluidic Devices.
The Yale journal of biology and medicine. 85. 501-21. The Yale journal of biology and medicine. 85. 501-21.

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Cell and Tissue Culture Cell and Tissue Culture

History of Cell Culture - Dr. Carrel’s Immortal History of Cell Culture - Dr. Carrel’s Flasks
Cells
Table 2. Pros and cons of Carrel’s flasks
Montrose Burrows (1884-1947) and Alexis Carrel (1873-1944) followed the
Harrison’s experiments. Carrel’s Flasks
They used different tissues of many species, and different types of culture media.
They obtained new culture from the old ones (continuous culture) without Pros Cons
establishing primary cell cultures from a new tissue.
• Longlived cultivation is possible • Control of lateral spread and
compared to hanging drop. coagulum of the plasma is
• Media-to-cell ratio is higher. difficult.
• Has improved gas exchange. • Variability in plasma coagulum
• Has larger capacity for cultures. affects the cell growth.
• Handy for biochemical studies. • Sample preservation and
histological preparations are
difficult to perform.
• Cultures in a single flask can be
Figure 19. Alexis Carrel’s glass culture flasks contaminated at the same time.

Carrel A. (1923). A METHOD FOR THE PHYSIOLOGICAL STUDY OF TISSUES IN VITRO. The Journal of experimental Millet, Larry & Gillette, Martha. (2012). Over a Century of Neuron Culture: From the Hanging Drop to Microfluidic Devices.
medicine, 38(4), 407–418. https://doi.org/10.1084/jem.38.4.407 The Yale journal of biology and medicine. 85. 501-21.

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Cell and Tissue Culture Cell and Tissue Culture

History of Cell Culture - Dr. Carrel’s Immortal
Cells
In January 1912 , first cell line was derived from chicken
embryo heart and maintained by washing with Ringer’s
solution and changing the medium.
After 2 years of the Carrel’s death, the cell line was
terminated.
Carrel defined the immortal cells that could live
indefinitely except for some lethal circumstances.
History of Cell Culture - Establishing Principles in
Cell Culture Maintaining
The significant impact on cell culture
development had started with the aseptic
techniques and tissue trypsinization
technique.

In 1956, Haff and Swim described cell aging in vitro.
They attributed cell aging to deficiencies in the culture
medium.
Figure 20. Alexis
Carrel. In 1938, Carrel published the book «The culture of
organs» that explains the cultivation techiques of whole
organs.
Alexis Carrel – Facts. NobelPrize.org. Nobel Prize Outreach AB 2023. Sun. 8 Oct 2023.
<https://www.nobelprize.org/prizes/medicine/1912/carrel/facts/>
In the early 20th century, Rous and Jones
developed the trypsinization technique,
including 3% trypsin solution.
Figure 21. Trypsinized chick
embryo cells from the study of
Rous and Jones.
Rous, P., & Jones, F. S. (1916). A METHOD FOR OBTAINING SUSPENSIONS OF LIVING CELLS FROM THE FIXED
TISSUES, AND FOR THE PLATING OUT OF INDIVIDUAL CELLS. The Journal of experimental medicine, 23(4), 549–555.
https://doi.org/10.1084/jem.23.4.549

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Media
History of Cell Culture - Cell Line Cultures’ History of Cell Culture - • Amino acids and

Development Cell Culture Conditions Labware vitamins

• Ions and trace elements
• Carbohydrate and
In 1948, the first immortalized cell line (the In 1951, the first human cell line (uterine organic supplements
Viscosity
«L» cell line) was derived from subcutaneous cervical cancer) was derived by Gay from In 1920s, Pannett and • Serum
Compton (1924), Gay (1936), • Antibiotics
mouse tissue by Earle. the patient named Henrietta Lacks. • Growth factors and
Table 3. Earliest derived cell lines Earle (1943), or Hanks (1948) hormones
salts were used as different
salt solutions in cell cultures. Cell Culture Potential
Osmolaritiy hydrogen
Environment (pH)
Between 1932 and 1962,
about 60 chemically defined
media were studied.
• Media199 developed by
Parker, Morgan, and Temperature Oxygen
Morton.
• Earle studied on protein Carbondioxide
free media for L cells.

Jedrzejczak-Silicka, M. (2017). History of Cell Culture. InTech. doi: 10.5772/66905

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Media Media
History of Cell Culture - • Amino acids and
History of Cell Culture - • Amino acids and

Cell Culture Conditions Cell Culture Conditions

Labware vitamins Labware vitamins
• Ions and trace elements • Ions and trace elements
• Carbohydrate and • Carbohydrate and
organic supplements organic supplements
Viscosity In 1940s, first effects of Viscosity
In 1955, Eagle developed • Serum • Serum

Basal Medium Eagle

• Antibiotics
• Growth factors and
(BME) for the L and HeLa hormones
antibiotics in in vitro cell • Antibiotics
• Growth factors and
culture was studied.
Herrel found that penicillin
hormones

cells. exhibits toxic action on mitosis

Potential Potential
4 years later, he developed Osmolaritiy
Cell Culture Osmolaritiy
Cell Culture
hydrogen due to impurities in hydrogen
Minimum essential Environment (pH) Environment (pH)
preparation.
medium (MEM) without Keilova worked on
non-essential amino acids. streptomycin.
In the same year, 1959, Lawrence found that some
Dulbecco and Freeman Temperature Oxygen concentrations of antibiotics Temperature Oxygen
causes respiratory damage or
developed Dulbecco’s necrotic changes and affects
modified MEM (DMEM). Carbondioxide
migration.
Carbondioxide

Krueger found the antibody

altering effect of streptomycin.
Yao, T., & Asayama, Y. (2017). Animal-cell culture media: History, characteristics, and current issues. Reproductive medicine and
biology, 16(2), 99–117. https://doi.org/10.1002/rmb2.12024

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Cell and Tissue Culture Cell and Tissue Culture

Media Media
History of Cell Culture - • Amino acids and
History of Cell Culture - • Amino acids and

Cell Culture Conditions Cell Culture Conditions

Labware vitamins Labware vitamins
• Ions and trace elements • Ions and trace elements
• Carbohydrate and • Carbohydrate and
In the 19th century, incubator was Viscosity organic supplements In 1909, W. K. Mulford Viscosity organic supplements
firstly used by Robert Koch. • Serum Pharmaceutical Co. presented the • Serum
Then, used by Virchow, Pasteur, • Antibiotics • Antibiotics
• Growth factors and first ventilated hood, which was • Growth factors and
Pettenkofer, Carrel, and Burrows. hormones designed to prevent infection with hormones
After 1960s, incubators with CO2 Mycobacterium tuberculosis during
support started to be used.
Potential the preparation of tuberculin. Potential
Today’s conditions are 5-10% CO2,
Osmolaritiy
Cell Culture Cell Culture
hydrogen In 1943, first publication explaining Osmolaritiy hydrogen
37℃ temperature, and 95% of Environment (pH) Environment (pH)
relative humidity.
microbiological cabinets was
published.
Van den Ende designed a safety cabinet
with electric furnace to create inward
airflow and to incinerate the exhausting
Temperature Oxygen Temperature Oxygen
air.
In 1960s, laminar flow clean room was
Carbondioxide developed. Carbondioxide
Today, cabins are equiped with HEPA
(high efficiency particle) filter and UV
light.
Figure 22. Incubator

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History of Cell Culture - Hayflick Phenomenon History of Cell Culture – Hayflick Phenomenon

Hayflick phenomenon or Hayflick limit

Dr. Witkowski presented three theories about Carrel’s immortal
represents the dividing number of the cells.
normal cell population before entering
senescence. 1 – Cell transformation theory
In 1961, Hayflick showed that normal
human fetal cells can divide 40-60 times 2 – Cell contamination theory
before losing the capacity of divide.
3 – Re-stocking theory
Phase I – The primary cells that
terminates with the formation of the first
confluent sheet.
Phase II – Abundant growth with many
Figure 23. Hayflick model
subcultivations.
Phase III – Cells stop dividing.

Hayflick, L., & Moorhead, P. S. (1961). The serial cultivation of human diploid cell strains. Experimental cell research, 25(3), 585-
621.

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Cell and Tissue Culture
History of Cell Culture - Immortality of HeLa
Cell Line
Henrietta Lacks was diagnosed with aggressive adenocarcinoma of the cervix by
Dr. Jones, in 1951. Samples from biopsy were send to Dr. George Gay
Firstly, it was noticed that the cells can be remained viable in chicken plasma.
Then, the cells were cultured in roller tubes and the cell line was called HeLa.
In 1952, Gay’s laboratory stated that the HeLa cell line was established and
maintained with continuous roller tube cultures for a year.
The cell line was distributed to the laboratories in the U.S. and other countries.
In 1991, Van Valen suggested that HeLa cells have become new species
«Helacyton gartleri» as a result of viral infections, numerous passages and
contaminations.
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Cell and Tissue Culture
History of Cell Culture – Immortalization of Cells
Hayflick defined the immortality term as a «life form capable of
indefinite survival in conditions where no changes have occured in
molecular composition from some arbitrary beginning».
In 50s, scientists concluded that cells exhibit contact inhibition of
growth. Rous sarcoma virus was the first evidence of cell’s
transformation by oncogenic retroviruses. The transformed cells
displayed the ability to continue proliferate even after they reached
confluence.
Simian virus 40 (SV40) was used for transformations.
In 1965, Harris and Watkins obtained the first hybrid mammalian
cells (human and mouse cells) via viral fusion.
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History of Cell Culture – First Monoclonal Antibodies History of Cell Culture – Phenomenon of Induced
Pluripotent Stem Cells (iPSCs)
In 1962, Gurdon replaced the nucleus of a frog
In 1984, Georges Köhler and Cesar egg with a nucleus from a mature frog cell. The
Milstein were produced monoclonal egg developed into a cloned frog.
antibodies by immortilizing the
In 2006, Yamanaka introduced 4 transcription
antibody producing cells. factors together and reprogrammed fibroblasts
into stem cells. Resulting cells could develop
into mature fibroblasts, gut cells, and nerve
cells.

Figure 30. iPSC generation

Figure 24. Figure 25. Figure 26. Figure 27. Production of a hybridoma
Niels K. Jerne Georges J. F. Cesar Milstein
Köhler Das, B. C., & Tyagi, A. (2014). Stem Cells: A Trek from Laboratory to Clinic to
Figure 28. Sir Figure 29. Shinya Industry. In Animal biotechnology (pp. 425-450). Academic Press.
Press release. NobelPrize.org. Nobel Prize Outreach AB 2023. Sun. 15 Oct 2023.
<https://www.nobelprize.org/prizes/medicine/1984/press-release/> John B. Gurdon Yamanaka
The Nobel Prize in Physiology or Medicine 1984. NobelPrize.org. Nobel Prize Outreach AB 2023. Sun. 15 Oct 2023. The Nobel Prize in Physiology or Medicine 2012. NobelPrize.org. Nobel Prize Outreach AB 2023. Sun. 15 Oct 2023.
<https://www.nobelprize.org/prizes/medicine/1984/summary/> <https://www.nobelprize.org/prizes/medicine/2012/summary/>

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History of Cell Culture – Future of Cell Cultures History of Cell Culture

Viral
vectors for
gene
therapy

3D Cell Vaccine To read more…

culture technology

https://www.intechopen.com/chapters/53566
Future
of Cell
Culture

Personal
therapy ?

Recombinant
protein
production

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Ece ÖZYAĞCI
Selay TORNACI
Ebru TOKSOY ÖNER
Marmara University, Department of Bioengineering

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