Derivation and cultivation of human tumor cells from primary tumors

Single-cell suspensions from human melanoma tissue were prepared using the Tumor Dissociation Kit in combination with the gentleMACS™ Dissociator. Single-cell suspensions contained 17% red blood cells (RBCs). To exclude the RBCs from subsequent flow cytometric analysis, the cells were labeled with CD235a (glycophorin A) antibody. This prevents overestimation of cell viability and absolute cell count due to RBCs that falsely appear in the PI^- fraction. Finally, viability amounted to 86% at a yield of 5.8×10⁷ cells per gram of tissue.

Ovarian carcinoma tumors were dissociated with the Tumor Dissociation Kit, human and the gentleMACS Octo Dissociator with Heaters. The depletion of non-tumor cells from the dissociated primary tumors was achieved with the Tumor Cell Isolation Kit, human. Purified untouched tumor cells were obtained, eliminating >95% of contaminating non-tumor cells (A). Untouched human tumor cell isolation from ovarian carcinoma pleural effusion samples containing low initial frequencies of tumor cells allowed for subsequent isolation of tumor subpopulations by MACS Technology as shown for the isolation of CD133⁺ cancer stem cells (B).

Cells were cultivated and propagated in vitro using Pancreas TumorMACS Medium directly after dissociation (A–C) and after tumor cell isolation (D). Morphological heterogeneity was preserved not only during initial passages but also in late passages when cell lines were derived from these cultures. The medium supported cell expansion both in adherent (A) and suspension cultures (B). This is of particular importance, as the data show that for some specimens even epithelial tumor cells grow better in a suspension culture of spheroid-like structures (B).

Cell line–derived xenografts fail to reflect the histological and functional features of primary human tumors since these cell lines show a homogeneous histology (A). Cell lines derived from primary human tumors and propagated in Pancreas TumorMACS Medium retain their initial heterogeneity. After xenotransplantation, these cell lines closely resemble the parental patient tumor in histological analyses (B).

Gene expression profiles of early (passage 5) and late (passage 15) passages of the respective cell lines were analyzed. High correlation coefficients clearly demonstrate that the global gene expression profile is maintained upon long-term cultivation in Pancreas TumorMACS Medium (A). Likewise, the high level of correlation of cell line–derived xenotransplanted tumors (DT) to the initial patient tumors (PT) shows the preservation of genetic features (B).