Primary cell cultures

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[12] Skloot R. (2010), The Immortal Life of Henrietta Lacks, New York City: Random ... Wonder woman: The life, death, and life after death of Henrietta Lacks, ...
Primary cell cultures – a useful tool or a dangerous trap? O. Michel1, D. Przystupski 1, N. Niedzielska 2, K. Bieżuńska-Kusiak 1, J. Saczko 1, J. Kulbacka1 1

Department of Medical Biochemistry, Wroclaw Medical University, Wroclaw, Poland 2 Department of Biomedical Engineering, Wroclaw University of Technology, Wrocław, Poland e-mail: [email protected]

The captivating history of cell cultures Justin Jolly (1870 –1953) The culture of the nucleated red blood cells of amphibians

Wilhelm Roux (1850 –1924) Maintaining the medullary plate of an embryonic chicken in a warm saline solution [4]

Robert Hook (1635–1702) Antonie van Leeuwenhoek (1632–1732) „If we reflect that a small creature such as this is provided, not only with external members, but also with intestines and other organs, we have no reason to doubt that a like creature, even if a thousand million times smaller, may already be provided with all its external and internal organs... though they may be hidden from our eyes.” [2]

Zacharias Jansen (1580 - 1638) Galileo Galilei (1564–1642) Invention of the first microscope


Alfred Vulpian (1826 – 1887) An attempt to culture tadpole cells

Ross G. Harrison (1870 –1959) Cultivation of the nerve fibers of a frog for several weeks in lymph fluid [5, 6].

Alexis Carrel (1873 -1944) "The cell is immortal. It is merely the fluid in which it floats that degenerates. Renew the fluid at regular intervals, give the cells what they require for nutrition, and as far as we know, the pulsation of life can go on forever.„ [7]

Sydney Ringer (1835-1910) „The solution composed of saline solution, with calcium chloride and potassium chloride is neutral, hence the ventricle will beat perfectly when supplied with a neutral fluid, and therefore alkalinity of the circulating fluid is not necessary for contractility.” [3]

The microscope designed by Galileo, most probably made by Giuseppe Campani [1].

Peyton Rous (1879-1970) The development of the trypsin solution in 1916

The outgrowth of the nerve fiber [6]. George Otto Gey(1899 –1970) Establishment of the HeLa cell line in 1951

2004 Hwang Woo-suk performs the first cloning of the human embryonic stem cells

2014 Haruko Obokata presents STAP method

Source: (last access: 27.02.18)

In January 2014 Haruko Obokata published two breakthrough articles in „Nature” in which she introduced a simple method of STAP (“stimulus-triggered acquisition of pluripotency”) [14,15]. Obokata revealed that „strong external stimuli such as a transient low-pH stressor reprogrammed mammalian somatic cells, resulting in the generation of pluripotent cells” [15]. However, soon after publication disturbing allegations emerged. It appeared that no other laboratory could reproduce this experiment. Eventually, genetic analysis showed that STAP cells were a regular embryonic stem cells taken from a freezer and relabelled

Haruko Obokata presenting her research results [16].

2006 Shinya Yamanaka discovers that mature cells can be reprogrammed to become pluripotent

Fritz Zernike (1888-1966) Construction of the first phase contrast microscope in 1941 [8]

Marvin Minsky (1927-2016) In 1957 creates a predecessor of today's widely used confocal laser scanning microscope

1940 Keilova, Cruikshank and Lowbury introduce antibiotics in tissue culture [8]

Alexis Carrel was undoubtedly the pioneer of tissue culture and its chief publicist. His practical contributions include among others the development of methods for collecting samples, describing techniques of cell cultures (such as passaging) and creating the specialized flasks in which cells could be maintained in sterilized conditions for a longer time. Regardless of this work, however, some of his achievements remain controversial. In 1912, Carrel established a culture from a chick embryo. Soon, he announced that cells can be cultured indefinitely [9]. In fact, he maintained the cultures for 34 years. The problem is, that no one else could obtain indefinite cell growth in vitro. In the mid-60s the dogma of cell immortality was finally overthrown [10]. So how was it possible for Carrel to keep his cultures for such a long period? The most probable explanation is that the culture was regularly contaminated with some fresh cells from incorrectly prepared embryo extract added to a medium. But is it really possible that the experienced A double arm Carrel flask with "12% liver" Nobel Prize Winner unintentionally committed such negligence? penciled on its sandblasted spot. [11]

Hwang Woo-suk was a professor at Seoul National University. In 2004 he announced in „Science” the creation of the human embryonic stem cells by cloning. Soon after publication, he was accused of severe ethical violations using eggs from his graduate students and from the black market. Shortly after, it emerged that much of his stem cell research had been faked. In fact, none of his 11 “cloned” stem cells matched their supposed donors [16]. Teratomas formed by human SCNT ES cells in the testes of SCID mice at 12 weeks after injection. Neuroepithelial rosset (A), pigmented retinal epithelium (B), ostoid island showing bony differentiation (C), cartilage (D), and glandular epithelium with smooth muscle and connective tissues [17]

Henrietta Lacks was the unwitting source of cells from a tumor biopsied during treatment for cervical cancer. These cells were then cultured by George Otto Gey who created the cell line known as HeLa, enabling the important breakthroughs in biomedical research. For example, by 1954, Jonas Salk was using HeLa cells in his research to develop the polio vaccine. Further it has been used in research into cancer, AIDS, the effects of radiation and toxic substances, gene mapping, and countless other scientific pursuits [12,13]. However, it is also the example of severe violation of patient’s rights - no consent was obtained to culture her cells, nor were she or her family compensated for their extraction or use. Yet, it is estimated that the total cell weight in the world exceeds 50 million tons

How to obtain a primary cell culture? The material is typically collected during a surgery or a biopsy. The tissue fragment is washed with sterile PBS buffer and placed in a plastic dish containing sterile culture medium It must then be immediately transferred to the laboratory under sterile conditions in a dedicated packaging. Next, it has to be fragmented. Different methods can be utilized to obtain tissue disintegration: chopping with scalpel

Pros and cons of primary cell cultures +

• Usually slow growth

• Biological reactions similar to in vivo conditions

• High costs of media and supplements

• Mirroring the conditions prevailing in a tissue

• High risk of contamination

• Reliable stress response

• Lack of precise characteristics

• Limitation of the animal experiments

• Limited lifespan

• Implementation of the personalized therapy

Dividing HeLa cells (© Dr. Thomas Deerinck / Visuals Unlimited / Corbis)

mechanical disaggregation

enzymatic disaggregation

For the enzymatic separation we can use for example the cold solution of trypsin or the preheated solution of collagenase. The way of disintegration as well as the culture medium is closely associated with the origin of a tissue. Once the culture is established we have to monitor cells, especially for attachment and the presence of blood cells. It is essential to freeze and examine each passage as heterogenous cultures frequently differentiate and change their properties over time.

• Differ depending on a sample and culture conditions • Bioethical issues

Conclusion We should learn our lesson from the history of other scientists. However, let’s not be discourage by these disturbing examples – after all, the mistakes by Obokato and Woo-suk were quickly caught by the vigilant scientists. Alexis Carrel once said: “All of us, at certain moments of our lives, need to take advice and to receive help from other people.” And so, we should maintain the meticulousness in research and do not hesitate to ask more experienced supervisors, reviewers and colleagues for a double-check. Then, we can effectively use our primary cells as a powerful tool for scientific research.

A graduał growth of the primary cell culture before (1-3) and after trypsynization (4-6).

References [1] Source: (last access 27.02.18).

[10] Hayflick L. "The limited in vitro lifetime of human diploid cell strains." Experimental cell research 37.3 (1965): 614-636.

[2] van Leeuwenhoek A: Letter to the Gentlemen of the Royal Society, 30 Mar 1685. In The Collected Letters of Antoni van Leeuwenhoek (1957), Vol. 5, 185.

[11] Source: (last access: 27.02.18)

[3] Ringer S. (1883). A further contribution regarding the influence of the different constituents of the blood on the contraction of the heart. The Journal of physiology, 4(1), 29-42.

[12] Skloot R. (2010), The Immortal Life of Henrietta Lacks, New York City: Random House, p. 2, ISBN 978-1-4000-5217-2

[4] Roux W. (1885) Beitriige zur entwicklungsmechanik des embryo. Z Biol.;21:411.

[13] Smith, V. (2002). Wonder woman: The life, death, and life after death of Henrietta Lacks, unwitting heroine of modern medical science. Baltimore City Paper, 17.

[5] Harrison RG, Greenman MJ, Mall FP, Jackson CM (1907). Observations of the living developing nerve fiber. Anat Rec. 1:116–128.

[14] Obokata H. et al. (2014) "Bidirectional developmental potential in reprogrammed cells with acquired pluripotency." Nature 505.7485: 676.

[6] Harrison RG(1910) The outgrowth of the nerve fiber as a mode of protoplasmic movement. Journal of Experimental Zoology 9:787-846.

[15] Obokata H. et al. (2014) "Stimulus-triggered fate conversion of somatic cells into pluripotency." Nature 505.7485:641.

[7] Carrel, A., & Rodker, J. (1935). Man, the unknown (p. 207). New York: Harper.

[16] Rasko, J., & Power, C. (2015). What pushes scientists to lie? The disturbing but familiar story of Haruko Obokata. The Guardian, 18.

[8] Jedrzejczak-Silicka M (2017). History of Cell Culture, New Insights into Cell Culture Technology, Dr. Sivakumar Joghi Thatha Gowder (Ed.), InTech, DOI: 10.5772/66905.

[17] Hwang, Woo Suk, et al. "Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst." Science 303.5664 (2004): 1669-1674.

[9] Carrel A. "Contributions to the study of the mechanism of the growth of connective tissue." Journal of Experimental Medicine 18.3 (1913): 287-298.

ACKNOWLEDGEMENTS: This work was supported by funds from the project STM.A040.17.039