Applications and Advances of Multicellular Tumor Spheroids: Challenges in Their Development and Analysis

Abstract

Biomedical research requires both in vitro and in vivo studies in order to explore disease processes or drug interactions. Foundational investigations have been performed at the cellular level using two-dimensional cultures as the gold-standard method since the early 20th century. However, three-dimensional (3D) cultures have emerged as a new tool for tissue modeling over the last few years, bridging the gap between in vitro and animal model studies. Cancer has been a worldwide challenge for the biomedical community due to its high morbidity and mortality rates. Various methods have been developed to produce multicellular tumor spheroids (MCTSs), including scaffold-free and scaffold-based structures, which usually depend on the demands of the cells used and the related biological question. MCTSs are increasingly utilized in studies involving cancer cell metabolism and cell cycle defects. These studies produce massive amounts of data, which demand elaborate and complex tools for thorough analysis. In this review, we discuss the advantages and disadvantages of several up-to-date methods used to construct MCTSs. In addition, we also present advanced methods for analyzing MCTS features. As MCTSs more closely mimic the in vivo tumor environment, compared to 2D monolayers, they can evolve to be an appealing model for in vitro tumor biology studies.

Keywords: 3D cell cultures; SPIM; cancer; microscopy; multicellular tumor spheroids (MCTS).

Figure 1

Characteristics of (a) mono-culture MCTS, (b) co-culture MCTS and (c) in vivo tumor.

Figure 2

Different scaffold-free methods for MCTS formation. (a) Liquid Overlay Technique, (b) Hanging drop assay and (c) Spinner cultures.

Figure 3

Scaffold-based methods for MCTS formation.

Figure 4

The alteration of the cell cycle stages from outer to inner layers of MCTS.

Figure 5

(a) In a confocal system the same objective is used to excite fluorophores and detect the emitted fluorescence. As the sample is scanned along the imaging plane, fluorophores bellow and above the imaging plane are also getting excited (blue cones of light). Since a pinhole is used to achieve optical sectioning, only photons emitted from the imaging plane (green cones of light) are finally detected by the photo-sensor; thus, unnecessary photobleaching and photodamage occurs. In case of a z-scan, each point of the sample is illuminated multiple times. (b) In a light-sheet based system, a thin “sheet” of light (blue light beam) is used to illuminate all fluorophores on the imaging plane simultaneously. Fluorescence from that plane is collected with an objective lens that is positioned orthogonally to the illumination axis and is detected using an array detector. In a case of z-scan, each plane is illuminated once, minimizing photobleaching and photodamage. (c) Profile of the illumination beam. The axial resolution in a light-sheet microscope is affected by the thickness of the illumination beam which is measured at the beam “waist”; the point where the beam width is smaller (denoted as 2w_0). The distance from the beam waist to the points where the beam width is equal to 2√2 w_0 is called Rayleigh range (z_R). The confocal parameter (b) is defined as the distance where the beam width is nearly homogeneous and is calculated as b=2z_R. The confocal parameter defines the effective FOV. (d) Beam profiles of light-sheets created with high or low numerical apertures (NA). Light-sheets created with high NA have thin beam waist and can be used to increase the axial resolution. However, the length in which the beam is homogeneous (b; confocal parameter) is short and the plane is illuminated heterogeneously. On the other hand, light-sheets created with low NA have wider beam waist and achieve lower axial resolution. However, their confocal parameter (b) is long enough to illuminate the whole plane homoge-neously. The thickness of a light-sheet should be selected that even illumination is achieved over the plane.