Calling attention to the use of false “endothelial” cell lines.

7 01 2010

To the Editor:

In the article authored by Berlado Andozia et al. published online 11 November 2009 (1), they used the cell line ECV304 as a “human endothelial cell line.”

For many years, ECV304 was reported as a spontaneously transformed line derived from a Japanese human umbilical vein endothelial cell (HUVEC) culture (2). However, a seminal report was published in 1999, indicating that ECV304 is a derivative of the human urinary bladder carcinoma T24 cell line because of cross-contamination. (3)

Cross-contamination has emerged as a real problem that seriously compromises the quality of research. In the late 1960s, a shocking report demonstrated that most (if not all) of the putative unique human cell lines available at that time were actually derivatives of the HeLa cell line. However, the problem is still expanding and affects many cell lines used as classical in vitro models for many years (4). The incidence of research papers flawed by the use of misidentified and cross-contaminated cell culture is estimated between 15% and 20%.

In regards to cross-contamination of ECV304, many efforts have been made by important cell and tissue repositories, including the German Culture Collection of Microorganisms and Cell Cultures, the first in reporting the cross-contamination; American Type Culture Collection, and the Japanese Collection of Research Bioresources, to spread the information that ECV304 is a derivative of T24. This information is explicitly stated in their catalogs and websites, and ATCC has even contacted all customers who purchased this cell line at anytime, alerting them about it.

However, in spite of these efforts, an increasing number of papers using ECV304 as “endothelial” cell line are published every year in different journals covering several biomedical research areas, and where endothelium plays an important role in both physiological and pathophysiological contexts.

Some authors have even argued its usefulness, considering some endothelial cell-like features displayed by this cell line [5].
However, researchers should seriously take into consideration that ECV304 is not of HUVEC origin, and therefore it is an inappropriate cell line to study endothelial cell biology.

As scientists, we view this situation with major concern; it must be faced up to with seriousness to stop the apparent lack of awareness of some researchers regarding the identity of this and other cell line(s).

Armando Rojas
Ileana Gonzalez
Hector Figueroa
Medicine Faculty
Catholic University of Maule
Talca, Chile

References
1. Beraldo M, Sales C, Franceschini SA, Torqueti MA, Silva MFm Ferriani RA. Ethinylestradiol and estradiol have different effects on oxidative stress and nitric oxide synthesis in human endothelial cell cultures. Fertil. Steril 2009. DOI: 10.1016/j.fertnstert.2009.08.052

2. Takahashi K, Sawasaki Y, Hata J, Mukai K, Goto T. Spontaneous transformation and immortalization of human endothelial cells. In Vitro Cell Dev Biol 1990; 26: 265–274.

3. Dirks WG MacLeod RAF Drexler HG. ECV304 (endothelial) is really T24 (bladder carcinoma): cell line cross- contamination at source. In Vitro Cell Dev Biol Anim. 1999; 35: 558-559.

4. Rojas A, Gonzalez I, Figueroa H. Cell line cross-contamination in biomedical research: a call to prevent unawareness. Acta Pharmacol Sin. 2008; 29:877-80.

5. Suda K, Rothen-Rutishauser B, Günthert M, Wunderli-Allenspach H. Phenotypic characterization of human umbilical vein endothelial (ECV304) and urinary carcinoma (T24) cells: endothelial versus epithelial features. In Vitro Cell Dev Biol Anim. 2001 ;37:505-14.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2010.01.006

The Authors Respond:

We appreciate the interest and the comments of Armando Rojas, Ileana Gonzalez, and Hector Figueroa about our study (1). Our study was the first designed in order to compare the endothelial effects of ethinylestradiol (EE) and of estradiol on nitric oxide (NO) production and protection against oxidative stress in human endothelial cell cultures.

As we mentioned in the manuscript, there are some limitations to all in vitro studies, ranging from the choice of the model to study endothelial function to the fact that in vitro findings do not always correspond to in vivo findings.

Human umbilical vein endothelial cells (HUVECs) have been used extensively as a model to study the human endothelial cell in vitro. HUVECs isolated from umbilical cords remain a readily available and popular source of endothelial cells. However, there are several disadvantages to the isolation and culture of these cells, including the risk of infection, exogenous growth factor requirement, and low proliferative capacity. An important feature of the endothelium is that its properties vary between different sites of the vasculature and from one moment in time to the next (2). These differences reflect the capacity of the endothelium to respond to the unique needs of the underlying tissue. Thus, the heterogeneity between HUVEC isolates from different cords can make critical interpretation of results difficult. Strictly speaking, even the primary cells (HUVECs) could represent a limitation as a model to study endothelial cell biology.

Morphological, immunochemical, and genetic studies on the ECV304 cell line (the model chosen in our study) have shown that this line is appropriate for use as a model system for angiogenesis in vitro and signal transduction (3,4). Despite the fact that genetic similarity between the two types of cell lines (ECV 304 and T24) has been reported by some researchers (5) but not by others (6), ECV 304 cells phenotypically show important endothelial characteristics not observed in T24 cells. ECV304 and T24 cells differ in growth behavior, cytoarchitecture, tight junction arrangement. transmembrane electrical resistance, and activity of gamma-glutamyltransferase. Several endothelial markers (von Willebrand factor, uptake of low-density lipoprotein, vimentin) could clearly be identified in ECV304, but not in T24 cells (4). In addition, ECV 304 cells produces NO (6,7) by activating endothelial nitric oxide synthase (8).

The ECV304 cell line exposes many endothelial features which, in view of the scarcity of suitable endothelial cell lines, still make it an attractive in-vitro model for endothelia (4). Furthermore, our results are in accordance with clinical studies that had already shown that EE and estradiol have different effects on endothelium function (9-15).

Although there is not any previous study testing our objective in HUVECs, other studies on NO synthesis upon hormone stimulation, using ECV 304 and HUVEC, had similar results (7,16). In conclusion, in the absence of an ideal model to study endothelial cell biology, ECV 304 can be used to evaluate endothelial characteristics that this line of cells presents.

Carolina Sales Vieira, M.D., Ph.D.a,c
Maria Regina Torqueti Tolloi, Ph.D.b
Rui Alberto Ferriani, M.D., Ph.D..a,c

aDepartment of Gynecology and Obstetrics
University of São Paulo Ribeirão Preto School of Medicine
bFaculty of Pharmacy of Ribeirão Preto University of São Paulo
cNational Institute of Hormones and Women’s Health
Ribeirão Preto, Brazil

References
1. Andozia MB, Vieira CS, Franceschini SA, Torqueti Tolloi MR, Silva de Sá MF, Ferriani RA. Ethinylestradiol and estradiol have different effects on oxidative stress and nitric oxide synthesis in human endothelial cell cultures. Fertil Steril. 2009 Nov 10. [Epub ahead of print]

2. Aird WC. Endothelial cell heterogeneity. Crit Care Med 2003; 31:S221–30.

3. Hughes SE. Functional Characterization of the Spontaneously Transformed Human Umbilical Vein Endothelial Cell Line ECV304: Use in an in Vitro Model of Angiogenesis. Exp. Cell Res. 1996; 225: 171-185.

4. Suda K, Rothen-Rutishauser B, Gunthert M, Wunderli-Allenspach, H., Phenotypic Characterization of Human Umbilical Vein Endothelial (ECV304) and Urinary Carcinoma (T24) Cells: Endothelial Versus Epithelial Features. In Vitro Cell Dev. Biol. Anim. 2002; 38: 185-6.

5. Dirks WG, MacLeod RAF, Drexler HG. ECV304 (endothelial) is really T24 (bladder carcinoma): cell line cross- contamination at source. In Vitro Cell Dev Biol Anim. 1999; 35: 558-9.

6. Gil’iano NIa, Semenova EG, Fedortseva RF, Konevega LV . Characteristics of the spontaneously transformed human endothelial cell line ECV304. II. Functional responses of the ECV304 cells. Cell and Tissue Biolog 2009; 3: 274-82.

7. Paulo M, Salvador MM, dos Anjos Neto Filho M, Montes MB, Franceschini SA, Toloi MR. Effectof isoflavone extracts from Glycine max on human endothelial cell damage and on nitric oxide production. Menopause 2009; 16: 539-544.

8. Jong Seon Park, Gu Ru Hong, Suk Whan Baek, Dong Gu Shin, Young Jo Kim, Bong Sup Shim. Expression and Regulation of Endothelial Nitric Oxide Synthase by Vascular Endothelial Growth Factor in ECV 304 Cells. J Korean Med Sci 2002; 17: 161-7.

9. de Kleijn MJ, Wilmink HW, Bots ML, Bak AA, van der Schouw YT, Planellas J et al. Hormone replacement therapy and endothelial function. Results of a randomized controlled trial in healthy postmenopausal women. Atherosclerosis 2001; 159: 357-65.

10. Guetta V, Quyyumi AA, Prasad A, Panza JA, Waclawiw M, and Cannon RO. The role of nitric oxide in coronary vascular effects of estrogen in postmenopausal women. Circulation. 1997; 96: 2795–2801.

11. de Kleijn MJ, Wilmink HW, Bots ML, Bak AA, van der Schouw YT, Planellas J et al. Hormone replacement therapy and endothelial function. Results of a randomized controlled trial in healthy postmenopausal women. Atherosclerosis 2001; 159: 357-65.

12. Gerhard M, Walsh BW, Tawakol A, Haley EA, Creager SJ, Seely EW et al. Estradiol therapy combined with progesterone and endothelium-dependent vasodilation in postmenopausal women. Circulation 1998; 98: 1158-63.

13. Merki-Feld GS, Imthurn B, Keller PJ. The effect of the menstrual cycle and of ethinylestradiol on nitric oxide, endothelin-1 and homocysteine plasma levels. Horm Metab Res. 2000; 32: 288-93.

14. John S, Jacobi J, Schlaich M, Delles C, Schmieder RE. Effects of oral contraceptives on vascular endothelium in premenopausal women. Am. J. Obstet Gynaec. 2000; 183: 28-33.

15. Lizarelli PM, Martins WP, Vieira CS, Soares GM, Franceschini SA, Ferriani RA, Patta MC. Both a combined oral contraceptive and depot medroxyprogesterone acetate impair endothelial function in young women. Contraception 2009; 79: 35-40.

16. Simoncini T, Fornari L, Mannella P, Caruso A, Garibaldi S, Baldacci C, Genazzani AR. Activation of nitric oxide synthesis in human endothelial cells by red clover extracts. Menopause 2005; 12: 69-77.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2010.01.005

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One response

28 01 2010
Armando Rojas

ECV304 cell line may even has some endothelial-like cell features, but authors using this cell line should not ever describe it as a “human endothelial cell line” because it does not. If authors chose this cell line as a their “model”, they should declare what it really is.
In regards to cell heterogeneity, it is a bias in any primary cell culture. However, in a tumoral cell line is very likely that this heterogeneity will be markedly higher than in any primary cell culture, due to genetic instability which is a very common feature of tumor cells. Additionally, the existance of heterogeneity in HUVECS, particularly that coming from different umbilical cords, probably may bring more strenght and significance to the results than that coming from any tumor cell line.

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