Three-dimensional culture of a mixed mullerian tumor of the ovary: expression of in vivo characteristics.

The Rotating-Wall Vessel (RWV) is a novel in vitro cell culture system used to successfully culture a cell line derived from a heterologous mixed mullerian tumor cell of the ovary. Although the original tumor was comprised of both epithelial and mesodermal components, long-term culture in conventional flasks established a cell line from this tumor with homogeneous epitheliallike growth characteristics (1). Cells from Passage 36 were seeded into a Rotating-Wall Vessel containing Cytodex-3 microcarrier beads. Scanning electron micrographs of tumor cells cultured for 32 d in the RWV showed the presence of heterogeneous cell populations organized into three-dimensional tissuelike architecture. Immunocytochemical analysis confirmed the cellular heterogeneity, as demonstrated by expression of both epithelial and mesenchymal antigens. Reverse transcription polymerase chain reaction amplification demonstrated the presence of mRNA for cellular oncogenes HER-2/neu, H-ras, K-ras, and tumor suppressor p53. Thus, there are two advantages to propagation of tissue in the RWV culture system:(a) tissue diversification representing populations present in the original tumor, and (b) the three-dimensional freedom to organize tissues morphologically akin to those observed in vivo. These data indicate that the RWV culture system is suitable for generating large quantities of ovarian tumor cells in vitro that are amenable to immunocytochemical, oncogenic, morphologic characteristics demonstrated in vivo.

Three-dimensional growth and differentiation of ovarian tumor cell line in high aspect rotating-wall vessel: morphologic and embryologic considerations.

Cancer of the ovary is the leading cause of death from gynecologic malignancy. To understand better these aggressive tumors, the development of in vitro models to study human ovarian cancer is critical. However, the establishment of long-term cell lines has been difficult, due to the generalized poor survival of patient tumor cells grown in primary culture. Satisfactory culture systems for ovarian tumor cells have therefore been limited. To study cellular interactions involved in the growth and differentiation of these tumors, a cell line was established from a mixed müllerian tumor of the ovary. This cell line, designated LN1, was cultured on microcarrier beads in the high aspect rotating-wall vessel. The tumor cells grown in this vessel readily proliferated without a requirement for cocultivation with a supportive cell layer. Evaluation of cellular morphology by phase contrast light microscopy and scanning electron microscopy revealed the presence of three-dimensional multicellular aggregates consisting of multiple cell-coated beads bridged together, as well as scattered aggregates of LN1 cells proliferating as spheroids free in suspension. In contrast to conventional culture systems, culture in the high aspect rotating-wall vessel facilitated the generation of multiple cell types that could be recovered. These results illustrate the ability of this culture system to provide the biological conditions necessary for pluripotent cell growth.

Reduced shear stress: a major component in the ability of mammalian tissues to form three-dimensional assemblies in simulated microgravity.

BHK-21 cells were cultured under various shear stress conditions in an Integrated Rotating-Wall Vessel (IRWV). Shear ranged from 0.5 dyn/cm2 (simulated microgravity) to 0.92 dyn/cm2. Under simulated microgravity conditions, BHK-21 cells complexed into three-dimensional cellular aggregates attaining 6 x 10(6) cells/ml as compared to growth under 0.92 dyn/cm2 conditions. Glucose utilization in simulated microgravity was reduced significantly, and cellular damage at the microcarrier surface was kept to a minimum. Thus, the integrated rotating wall vessel provides a quiescent environment for the culture of mammalian cells.