Date Published: June 11, 2018
Publisher: Public Library of Science
Author(s): Bartłomiej Taciak, Maciej Białasek, Agata Braniewska, Zuzanna Sas, Paulina Sawicka, Łukasz Kiraga, Tomasz Rygiel, Magdalena Król, David D. Roberts.
Established cell lines are widely used in research, however an appealing question is the comparability of the cells between various laboratories, their characteristics and stability in time. Problematic is also the cell line misidentification, genetic and phenotypic shift or Mycoplasma contamination which are often forgotten in research papers. The monocyte/macrophage-like cell line RAW 264.7 has been one of the most commonly used myeloid cell line for more than 40 years. Despite its phenotypic and functional stability is often discussed in literature or at various scientific discussion panels, their stability during the consecutive passages has not been confirmed in any solid study. So far, only a few functional features of these cells have been studied, for example their ability to differentiate into osteoclasts. Therefore, in the present paper we have investigated the phenotype and functional stability of the RAW 264.7 cell line from passage no. 5 till passage no. 50. We found out that the phenotype (expression of particular macrophage-characteristic genes and surface markers) and functional characteristics (phagocytosis and NO production) of RAW 264.7 cell line remains stable through passages: from passage no. 10 up to passage no. 30. Overall, our results indicated that the RAW 264.7 cell line should not be used after the passage no. 30 otherwise it may influence the data reliability.
Established cell lines are widely used all over the world for both in vitro and in vivo analyses. The reason for that is unlimited source of cells with similar genotype and phenotype through consecutive passages. However, in recent years authors are more cautious in interpretation of data obtained from experiments conducted only on the established cell lines. The most appealing question is obviously the characteristics of the cells and their comparability and stability between various laboratories and even various passages . Cell line misidentification, Mycoplasma contamination or genotypic and phenotypic shift when cultured for prolonged time, are the important issues very often missed in research papers. The immortalized cell lines are usable through many passages but their increasing number of passages enhances probability of genotype and phenotype change . In most of the research papers (even these published in top journals) there is no information about the passage number of the used cell lines or phenotype characteristics. The use of many cell lines has been questioned suggesting superiority of primary cells over the established cell lines [3–6]. Comparison of cells of the lower passages with cells of the higher passages being continuously in culture, demonstrated significant changes in their morphology, growth and differentiation ability with their consecutive passages [7,8]. From this point of view, macrophages are particularly interesting cells. They are famous for their plasticity and possibility to polarization and differentiation to osteoclasts, Kuppfer cells or dendritic cells [9–11]. Therefore, macrophages are a very diverse cell type. They are found in every organ and tissue in the body, however their phenotype is very different depending on the physiological state . Macrophages are very sensitive to environmental conditions and are able to quickly adapt to new stimuli . Depending on the type of activation manifested by produced cytokines and expression of surface markers, macrophages are classified as the M1 or M2 subgroups. The M1 macrophages (iNOS+, CD80+, MHCII+), classically activated (e.g. by LPS or IFN-γ) are pro-inflammatory cells, secreting following cytokines: IFN-γ and TNF-α. On the other hand, anti-inflammatory M2 macrophages (Arg-1+, CD163+, CD206+) are induced by IL-4 or IL-10 [14,15].
The RAW 264.7 cells are often used for in vitro and in vivo studies as an appropriate monocyte/macrophage model . However, their stability between various laboratories and passages is questionable . The ATCC, the main supplier of the cell lines, recommends their use till passage no. 18, however without showing any strong rationale for this .