Introduction

In our study, we aim to present a primary culture model of renal cell carcinoma using an organoid culture method which preserves primary tissue characteristics.

Methods

Renal cell carcinoma tissues were obtained from advanced cancer patients and were degraded through 1 hour of incubation at 37? using collagenase. Separated cells were mixed with matrigel (3D culture) or applied on top of a large amount of previously harden matrigel (3D on-top culture). The cells were replaced with fresh medium containing EGF, Hydrocortisone, insulin, Noggin, and Leucin in DMEM/F12 every 2 days. The cells grown in general cell culture dish were used as a control after being subcultured 4 times. To investigate whether the original characteristics of the primary cancer were preserved, the expressions of carbonic anhydrase IX (CA9), vimentin (VIM), and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP-3) were compared using immunohistochemistry, western blotting, and confocal microscopy.

Results

Early stage renal cell carcinoma contained abundant clear cytoplasm within the cells. After being subcultured, in the cells grown in the normal culture dish, clear cytoplasm was dramatically reduced, and the size of nuclear and cytoplasm was increased, while the dendritic change was observed (Fig. 1A). However, the initial appearance of renal cell carcinoma was maintained in the 3D culture and 3D on-top culture incubated over the same period (Fig. 1B). Moreover, the expression for CA9, VIM and BNIP-3, which are the major genes for renal cell carcinoma, are also well-maintained compared to the control group.

Conclusions

In this study, we present a new primary 3D culture method appropriate for cancer cells using a modification of the pre-existing organoid culture method. This culture is characterized by its preservation of primary tumor characteristics in cancer tissue through progenitor cell growth. The methodology described here should enable the procurement of cells which maintain primary cancer properties, a major issue in personalized medicine research. Furthermore, it can be presented as a new alternative to replace the patient-derived xenograft model in the new anti-cancer drug selection stage which offers advantages regarding both time and cost.

Funding

none