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Solid tumors grow in a three-dimensional (3D) spatial conformation, resulting in a heterogeneous exposure to oxygen and nutrients as well as to other physical and chemical stresses. To mimic the 3D spatial conformation, 3D culture models have been used in cancer research since the diffusion-limited distribution of oxygen (hypoxia), nutrients, metabolites, and signaling molecules is not mimicked in conventional two-dimensional (2D) monolayer cultures. Cancer stem cell (CSCs) are defined as a small subset of cells within a tumor with the ability to self-renew and often drive tumor progression and recurrence after chemotherapy treatment. Traditionally, cancer stem cells have been isolated from cancer cell lines and tumor biopsies and grown in undefined serum containing media using 3D tumorsphere cultures. Here we report a new defined serum-free cancer stem cell media that supports the expansion of human prostate (E006AA) and breast (MCF-7) cancer cell lines using a 3D tumorsphere culture method. Both cells showed continuous proliferation supported during serial passage of 3D tumorsphere cultures. In addition to the stable proliferation, increased Aldehyde Dehydrogenase (ALDH) expressing cancer stem cell populations were observed after multiple passages in this new 3D cancer stem cell media.
Tumorsphere Formation and Expansion
Cancer Stem Cell Identification
Cancer stem cell express elevated levels of Aldehyde Dehydrogenase (ALDH). AldeRed™ ALDH Detection Assay enables live cancer stem cell identification and isolation based on aldehyde dehydrogenase (ALDH) expression using a red-shifted fluorescent substrate for ALDH, allowing concurrent use of green fluorescent reporters including GFP cell lines.
Figure 1.Stable Growth of Cancer Cells in 3dGRO™ Spheroid Medium. Cumulative population doublings of MCF7 cells and E006AA cells during serial passage of 3D tumorsphere cultures was plotted.
Figure 2.Tumorsphere formation of MCF7 Breast Cancer Cells at Passage 1 (A) and Passage 5 (B) cultured in 3dGRO™ Spheroid Medium.
Figure 3.Tumorsphere formation of E006AA Prostate Cancer Cells at Passage 1 (A) and Passage 5 (B) cultured in 3dGRO™ Spheroid Medium.
Figure 4.An enrichment of ALDHhigh cancer stem cell populations occurs when cancer cells are grown in 3dGRO™ Spheroid Medium after 5 passages in 3D tumorsphere cultures.