The inventor of tissue culture

American scientist Ross Granville Harrison from Yale University is

credited as being the first researcher to succeed at developing animal tissue

cultures outside of the body.3

The discovery of HeLa cells

Cancerous tissue samples are taken from Henrietta Lacks, a woman

diagnosed with an aggressive form of cervical cancer, whom later that year died at 31 years of age. The cancer cells collected during Lacks’ time in

hospital became the first human cancer cell line, known today as HeLa.5,6

Raji cell line established

The first continuous hematopoietic cell line was derived from a patient

diagnosed with Burkitt’s lymphoma.8

Cross-contamination identified in culture

The first case of cross-contamination was reported in Science in 1981 by

Walter Nelson-Rees and colleagues.10

Cell line authentication via DNA fingerprinting

Dennis Gilbert and colleagues develop a method for the identification and

individualization of human cells using DNA fingerprinting.13

UKCCCR guidelines for the use of cell lines in

cancer research published

UKCCCR guidelines are developed to highlight potential problems with cell

culture “and provide recommendations as to how they may be identified,

avoided or where possible eliminated.”16

Growth of tumor tissues in vitro

Alexis Carrel and Montrose T. Burrows generate hundreds of cultures derived from Rous chicken sarcoma.4

Mycoplasma identified in culture

Johns Hopkins’ researchers Lucille Robinson, Ruth Wichelhausen and Bernard Roizman report the discovery of mycoplasma in infected HeLa cultures.7

K562 cell line established

The immortalized K562 cell line was derived from a 53-year-old Caucasian female patient diagnosed with chronic myelogenous leukemia.9

NCI60 and anticancer drug screening

The National Cancer Institute (NCI) developed the NCI60 human tumor cell line for anticancer drug screening, until this point in time, the selection of drug compounds for further development had relied predominantly on in vivo models.11,12

Key guideline established

Hans G. Drexler and Yoshinobu Matsuo published a guideline for the characterization and publication of human malignant hematopoietic cell lines and reported on the importance of using well-characterized human B cell precursor leukemia cell lines.14,15

Evading apoptosis

Metastasis

Angiogenesis

Immunology

LYMPH VESSEL

BLOOD VESSEL

References

1. Gillet J-P, Varma S, Gottesman MM. The clinical relevance of cancer cell lines. J Natl Cancer Inst. 2013;105(7):452-458. doi: 10.1093/jnci/djt007

2. Ravi M, Ramesh A, Pattabhi A. Contributions of 3D cell cultures for cancer research. J Cell Physiol. 2017;232(10):2679-2697. doi: 10.1002/jcp.25664

3. Abercrombie M. Ross Granville Harrison, 1870-1959. Biographical Memoirs of Fellows of the Royal Society. 1961;7:110-126. doi:10.1098/rsbm.1961.0009

4. Carrel A, Burrows MT. Cultivation in vitro of malignant tumors. J Exp Med. 1911;13(5):571-575. doi:10.1084/jem.13.5.571

5. MacDonald A. Henrietta Lacks: The Mother of Modern Medicine. Technology Networks. https://www.technologynetworks.com/cell-science/articles/henrietta-lacks-the-mother-of-modern-medicine-303228 Published May 23, 2018. Accessed March 25, 2021

6. MacDonald A. 5 Contributions HeLa Cells Have Made to Science. Technology Networks . https://www.technologynetworks.com/cell-science/lists/5-contributions-hela-cells-have-made-to-science-305036 Published June 13, 2018. Accessed March 25, 2021

7. Robinson LB, Wichelhausen RH, Roizman B. Contamination of human cell cultures by Pleuropneumonialike organisms. Science. 1956;124(3232):1147-1148. doi: 10.1126/science.124.3232.1147

8. Karpova MB, Schoumans J, Ernberg I, Henter J-I, Nordenskjöld M, Fadeel B. Raji revisited: cytogenetics of the original Burkitt’s lymphoma cell line. Leukemia. 2005;19(1):159-161. doi: 10.1038/sj.leu.2403534

9. Lozzio CB, Lozzio BB. Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood. 1975;45(3):321-334. doi: 10.1182/blood.V45.3.321.321

10. Nelson-Rees WA, Daniels DW, Flandermeyer RR. Cross-contamination of cells in culture. Science. 1981;212(4493):446-452. doi: 10.1126/science.6451928

11. Shoemaker RH. The NCI60 human tumour cell line anticancer drug screen. Nat Rev Cancer. 2006;6(10):813-823. doi: 10.1038/nrc1951

12. Boyd MR. The NCI In Vitro Anticancer Drug Discovery Screen. In: Teicher BA, ed. Anticancer Drug Development Guide: Preclinical Screening, Clinical Trials, and Approval. Cancer Drug Discovery and Development. Humana Press; 1997:23-42. doi: 10.1007/978-1-4615-8152-9_2

13. Gilbert DA, Reid YA, Gail MH, et al. Application of DNA fingerprints for cell-line individualization. Am J Hum Genet. 1990;47(3):499-514. PMCID: PMC1683851

14. Drexler HG, Matsuo Y. Guidelines for the characterization and publication of human malignant hematopoietic cell lines. Leukemia. 1999;13(6):835-842. doi: 10.1038/sj.leu.2401428

15. Matsuo Y, Drexler HG. Establishment and characterization of human B cell precursor-leukemia cell lines. Leukemia Research. 1998;22(7):567-579. doi: 10.1016/S0145-2126(98)00050-2

16. UKCCCR guidelines for the use of cell lines in cancer research. Br J Cancer. 2000;82(9):1495-1509. doi: 10.1054/bjoc.1999.1169

17. Boussommier-Calleja A. The fine cell line. Technology Networks. https://www.technologynetworks.com/cancer-research/articles/the-fine-cell-line-326232 Published Oct 31, 2019. Accessed March 24, 2021.

18. Namekawa T, Ikeda K, Horie-Inoue K, Inoue S. Application of prostate cancer models for preclinical study: advantages and limitations of cell lines, patient-derived xenografts, and three-dimensional culture of patient-derived cells. Cells. 2019;8(1):74. doi: 10.3390/cells8010074

19. Chaicharoenaudomrung N, Kunhorm P, Noisa P. Three-dimensional cell culture systems as an in vitro platform for cancer and stem cell modeling. World J Stem Cells. 2019;11(12):1065-1083. doi: 10.4252/wjsc.v11.i12.1065

20. Miserocchi G, Mercatali L, Liverani C, et al. Management and potentialities of primary cancer cultures in preclinical and translational studies. J Transl Med. 2017;15(1):229. doi: 10.1186/s12967-017-1328-z

21. Brown HK, Schiavone K, Tazzyman S, Heymann D, Chico TJ. Zebrafish xenograft models of cancer and metastasis for drug discovery. Expert Opin Drug Discov. 2017;12(4):379-389. doi: 10.1080/17460441.2017.1297416

22. Kapałczyńska M, Kolenda T, Przybyła W, et al. 2D and 3D cell cultures – a comparison of different types of cancer cell cultures. Arch Med Sci. 2018;14(4):910-919. doi: 10.5114/aoms.2016.63743

23. Nakanuma Y, Katayanagi K, Kawamura Y, Yoshida K. Monolayer and three-dimensional cell culture and living tissue culture of gallbladder epithelium. Microsc Res Tech. 1997;39(1):71-84. doi: 10.1002/(SICI)1097-0029(19971001)39:1<71::AID-JEMT6>3.0.CO;2-2

24. Heller A, Angelova AL, Bauer S, et al. Establishment and characterization of a novel cell line, ASAN-PaCa, derived from human adenocarcinoma arising in intraductal papillary mucinous neoplasm of the pancreas. Pancreas. 2016;45(10):1452-1460. doi: 10.1097/MPA.0000000000000673

25. Antoni D, Burckel H, Josset E, Noel G. Three-dimensional cell culture: a breakthrough in vivo. Int J Mol Sci. 2015;16(3):5517-5527. doi: 10.3390/ijms16035517

26. Costa EC, Moreira AF, de Melo-Diogo D, Gaspar VM, Carvalho MP, Correia IJ. 3D tumor spheroids: an overview on the tools and techniques used for their analysis. Biotechnol Adv. 2016;34(8):1427-1441. doi: 10.1016/j.biotechadv.2016.11.002

27. Huch M, Knoblich JA, Lutolf MP, Martinez-Arias A. The hope and the hype of organoid research. Development. 2017;144(6):938-941. doi: 10.1242/dev.150201

28. Jensen C, Teng Y. Is it time to start transitioning from 2D to 3D cell culture? Front Mol Biosci. 2020;7. doi: 10.3389/fmolb.2020.00033

29. Boussommier-Calleja, A. How To Prevent Cell Culture Contamination. Technology Networks. https://www.technologynetworks.com/tn/how-to-guides/how-to-prevent-cell-culture-contamination-htg-299231. Published March 29, 2018. Accessed March 25, 2021.

30. Savelieff, MG. Cancer Cell Line Choice: 3 Considerations to Make. Technology Networks. https://www.technologynetworks.com/cancer-research/lists/cancer-cell-line-choice-3-considerations-to-make-309837 Published March 29, 2018. Accessed March 25, 2021.

31. Nims RW, Sykes G, Cottrill K, Ikonomi P, Elmore E. Short tandem repeat profiling: part of an overall strategy for reducing the frequency of cell misidentification. In Vitro Cell Dev Biol Animal. 2010;46(10):811-819. doi: 10.1007/s11626-010-9352-9

The value of cell culture in cancer researchCell culture is an invaluable tool for cancer research, used in studies to improve our understanding of fundamental cancer biology and as preclinical models to develop and test drugs.

For several years, cell lines have helped uncover many intricacies of cancer pathogenesis,1 providing a simple, low cost and rapid way to test hypotheses.

Cancer cell line breakthroughs

The development of increasingly advanced 3D platforms is enabling researchers to build models that more closely mimic the in vivo environment and that offer greater predictive power.2

In this infographic, we take a closer look at some of the key cancer cell line breakthroughs, explore the value of primary cultures and 3D models in cancer research and highlight tips and tricks for cancer researchers to help them avoid common errors and issues when culturing cells.

for CancerResearch

Human primary cells or immortalized cancer cell lines? The development of immortalized cell lines provided scientists with easy access to large numbers of cells to use in their experiments, driving forward research in cancer and other disciplines. However, the limitations of immortalized cell lines and the advantages that primary cells offer are increasingly being recognized.17,18

Why choose 3D?3D cultures can help to bridge the gap25 between animal models and conventional, monolayer 2D cultures and show promise for addressing the issue of high drug attrition rates in cancer.

Immortalized cancer cell lines

Implant

Primary cells

Infinite lifespan

Altered morphology and genomic content, lose cell specific characteristics

Low physiological relevance

Lower cost

Easy to maintain – requires standard culture conditions

Cells from single donor result in cell uniformity

Unlimited availability

Finite lifespan

Healthy morphology, genetically stable, retain cell identity

High physiological relevance

Higher cost

Difficult to maintain – each cell type requires specific

culture conditions

Donor variability leads to cells with variable characteristics

Limited availability

The value of primary cultures in cancer research

Tissue Sample

Primary Culture

Murine Model

Xenograft

Zebrafish Model

3D Culture SystemsCancer cell characteristics can be closely mimicked using 3D cell models. 3D culture techniques include:19

• Liquid overlay

• Hanging drop

• Hydrogel embedding

• Scaffolds

• Spin flask bioreactor

• Bioprinting

• Microfluidics

• Low adhesion plates

Cell Line Establishment It may be necessary to establish a new cell line if there is no commercial option for the type of cancer or stage of disease being studied.24

Spheroids26

• Spherical cellular aggregates grown without scaffolding.

• Provide useful models of the in vivo microenvironment of tumors.

Organoids27

• Tiny self-organizing 3D assemblies of cells that exhibit both the structural resemblance and functional characteristics of a specific organ.

• Used to model a wide range of tumors including brain, lung, breast and colon.

Bioreactor Bioreactor systems can be used to regulate a cell culture’s physiological properties via controlled fluid perfusion.20

Primary cells can be transplanted into a model organism to monitor processes that are difficult to recapitulate in vitro (e.g., angiogenesis and metastasis).20,21

Monolayer CultureCells in a monolayer come into contact with the culture vessel, neighboring cells and the culture medium. Specific biological features of cultured cells are gradually lost after several passages.22,23

3D culture offers several advantages:28

Greater physiological relevance

Better reproduce the cellular microenvironment, including nutrient, oxygen, waste and drug gradients

Avoid ethical implications of animal models

Enable higher-throughput screening than animal models

Key considerations29,30,31

Choose the right cell line for your research

Culture the cells without introducing contaminants

Consider what growth conditions your cells require to thrive

Confirm the authenticity of your culture before experimenting

Carry out your analysis

Cryopreserve your cells

Is the cell line capable of answering the hypothesis you set forward?

Does the cell line possess the proteins, genes or therapeutic targets you are investigating?

Cell cultures can become contaminated by biological and/or chemical contaminants.

However, the risk of contamination can be minimized by doing the following:

• Wear the correct personal protective equipment

• Set up your culture hood correctly and spray everything with ≥ 70% ethanol or industrial methylated spirits

• Review material safety data sheets and follow standard operating procedures

• Use sterile reagents and labware, open sterile packets inside the hood

• Clean incubators and water baths regularly

• Dispose of contaminated samples correctly

What is the preferred growth media, temperature, CO2 level, confluency, culture plate matrix?

Caution: Phenotypic drift can occur if cell lines are not cultured in the correct media.

Check the morphology of your cells under the microscope

Authenticate using short tandem repeat (STR) profiling

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1

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5

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Metabolism

Tumor morphology Drug discovery and toxicology