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. 2017 Aug;69(4):565-578.
doi: 10.1007/s10616-016-0063-2. Epub 2017 Mar 20.

Colony, hanging drop, and methylcellulose three dimensional hypoxic growth optimization of renal cell carcinoma cell lines

Damian Matak  1   2 , Klaudia K Brodaczewska  1 , Monika Lipiec  1   3 , Łukasz Szymanski  1   4   5 , Cezary Szczylik  1 , Anna M Czarnecka  6
Affiliations

Colony, hanging drop, and methylcellulose three dimensional hypoxic growth optimization of renal cell carcinoma cell lines

Damian Matak et al. Cytotechnology. 2017 Aug.

Abstract

Renal cell carcinoma (RCC) is the most lethal of the common urologic malignancies, comprising 3% of all human neoplasms, and the incidence of kidney cancer is rising annually. We need new approaches to target tumor cells that are resistant to current therapies and that give rise to recurrence and treatment failure. In this study, we focused on low oxygen tension and three-dimensional (3D) cell culture incorporation to develop a new RCC growth model. We used the hanging drop and colony formation methods, which are common in 3D culture, as well as a unique methylcellulose (MC) method. For the experiments, we used human primary RCC cell lines, metastatic RCC cell lines, human kidney cancer stem cells, and human healthy epithelial cells. In the hanging drop assay, we verified the potential of various cell lines to create solid aggregates in hypoxic and normoxic conditions. With the semi-soft agar method, we also determined the ability of various cell lines to create colonies under different oxygen conditions. Different cell behavior observed in the MC method versus the hanging drop and colony formation assays suggests that these three assays may be useful to test various cell properties. However, MC seems to be a particularly valuable alternative for 3D cell culture, as its higher efficiency of aggregate formation and serum independency are of interest in different areas of cancer biology.

Keywords: Colony; Hanging drop; Hypoxia; Methylcellulose; RCC.

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Figures

Fig. 1
Fig. 1
The process of compact aggregate creation. Changes in 786-O morphology under normoxic conditions in time—hanging drop assay. Magnification ×40
Fig. 2
Fig. 2
Differences in aggregation potential of various cell lines; cells with higher potential to aggregate showed reduced time. Caki-2, ACHN, and ASE-5063 were not able to create aggregate
Fig. 3
Fig. 3
Morphology of ACHN and ASE-5063 cells in normoxia after the 12th day of hanging drop culture. Magnification ×40
Fig. 4
Fig. 4
Semi-soft agar colonies of RCC cell lines in different oxygen partial pressures. Magnification ×40
Fig. 5
Fig. 5
MC aggregates at different time lapses. Magnification ×40
Fig. 6
Fig. 6
The influence of MC concentration on cellular aggregation. Magnification ×40
Fig. 7
Fig. 7
The influence of FBS concentration on cellular aggregation. Magnification ×40
Fig. 8
Fig. 8
The influence of oxygen on cellular aggregation. Magnification ×40
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