Formation of stable small cell number three-dimensional ovarian cancer spheroids using hanging drop arrays for preclinical drug sensitivity assays

Abstract

Background Ovarian cancer grows and metastasizes from multicellular spheroidal aggregates within the ascites fluid. Multicellular tumor spheroids are therefore physiologically significant3Din vitro models for ovarian cancer research. Conventional hanging drop cultures require high starting cell numbers, and are tedious for long-term maintenance. In this study, we generate stable, uniform multicellular spheroids using very small number of ovarian cancer cells in a novel 384 well hanging drop array platform. Methods We used novel tumor spheroid platform and two ovarian cancer cell lines (A2780 and OVCAR3) to demonstrate the stable incorporation of as few as 10 cells into a single spheroid. Results Spheroids had uniform geometry, with projected areas (42.60 × 103 μm–475.22 × 103 μm2 for A2780 spheroids and 37.24 × 103 μm2–281.01 × 103 μm2 for OVCAR3 spheroids) that varied as a function of the initial cell seeding density. Phalloidin and nuclear stains indicated cells formed tightly packed spheroids with demarcated boundaries and cell–cell interaction within spheroids. Cells within spheroids demonstrated over 85% viability. 3D tumor spheroids demonstrated greater resistance (70–80% viability) to cisplatin chemotherapy compared to 2D cultures (30–50% viability). Conclusions Ovarian cancer spheroids can be generated from limited cell numbers in high throughput 384 well plates with high viability. Spheroids demonstrate therapeutic resistance relative to cells in traditional 2D culture. Stable incorporation of low cell numbers is advantageous when translating this research to rare patient-derived cells. This system can be used to understand ovarian cancer spheroid biology, as well as carry out preclinical drug sensitivity assays.

Self-Renewal and CSCs In Vitro Enrichment: Growth as Floating Spheres

“…We have previously demonstrated that by utilizing hanging drop plates, which provide a 3D environment to culture few to single cells at a time, we can recapitulate the key features of the innate tumor microenvironment. The hanging drop method has been found to more closely mimic the outcomes of drug success in vivo, providing a more accurate representation of effectiveness and patient response [9–11]. …”

Review: Mechanotransduction in ovarian cancer: Shearing into the unknown

“…With this combinatory approach, it will be possible to gain a more complete picture of the cancer microenvironment and ascertain potential avenues of treatment. Additionally, non-cell factors such as chemotaxis, 130 3D culture 94,[131][132][133][134][135][136] , and hypoxia 137 should be considered for future investigations, in conjunction with mechanical cues to create a microenvironment that can more fully recapitulate in vivo conditions. The study of ovarian cancer mechanotransduction promises to improve patient treatment through future investigations that utilize designs pertinent to the specific microenvironment.…”

Biodynamic imaging for phenotypic profiling of three-dimensional tissue culture

“…Several methods are used to generate 3-D multicellular tumor spheroids including: (i) Bioreactors(BRs) in which cells or spheroids aggregate in the chamber; [38][39][40] (ii) nonadherent plates that prevent attachment; 41,42 (iii) hanging-drop (HD) plates where cells are grown in a suspended droplet to form one single spheroid; [43][44][45][46] (iv) multicellular layers; [47][48][49] and (v) organoid culture 50,51 among others. In this paper, BDI was used to assess the different cellular responses to paclitaxel of cancer spheroids obtained by using the three most common approaches: BR, HD, and nonadherent plate growth.…”