3D cell culture: spheroids
Spheroids are three-dimensional aggregates of cells that form when the cells are seeded onto an ultra-low attachment (ULA) surface and are thus forced to adhere to each other. Spheroids represent a more accurate model of physiologically relevant cell density as compared to the 2D cell culture. It also means that nutrients, oxygen, and other compounds of interest added to the spheroid-surrounding environment cannot reach the cells inside the spheroid as quickly and efficiently as in case of cells grown on the adherent surfaces. However, not all cell lines or primary cells form spheroids readily and the phenotype of spheroid (circularity, area, etc) can be affected by the cell culture medium and other treatment conditions.
We have so far succeeded in preparation of spheroids from the following cell lines:
· Non-small cell lung cancer – A549;
· Glioblastoma – U-251 MG, U-87 MG, T98-G;
· Ovarian cancer – SKOV3;
· Breast cancer – MCF7;
· Immortalized endometrial stromal fibroblasts – St-T1b, T-HESC.
We have so far succeeded in preparation of spheroids from the following cell lines:
· Non-small cell lung cancer – A549;
· Glioblastoma – U-251 MG, U-87 MG, T98-G;
· Ovarian cancer – SKOV3;
· Breast cancer – MCF7;
· Immortalized endometrial stromal fibroblasts – St-T1b, T-HESC.
Video 1. Time-lapse imaging of spheroid formation from U-251 MG cells after seeding onto ULA plate. Images were taken with 30 min intervals using Cytation 5 BioTek microscope (automated focussing). The time-scale in hours is shown in the top left corner and the spheroid size scalebar in the bottom right corner.
On average, seeding of 1000 to 10000 cells per well of a 96-well plate is a suitable starting point for formation of a single spheroid per well; after 48 h post-seeding, the first round of imaging can be performed. The imaging can be repeated every 24 h for even up to two weeks, provided that the medium is exchanged after every 48 h-72 h. As spheroids are usually ca 300-600 microns in diameter, 4× objective is sufficient to capture a bright-field image.
Figure 1. Dependence of spheroid diameter on the number of seeded cells at 24 h or 72 h post-seeding (quantified using ImageJ). Both cell lines were grown in phenol-red containing Eagle's Minimum Essential Medium supplemented with 10% FBS and antibiotic/antimycotic mixture. Note that the spheroid size does not increase in time a least during the first days on the ULA plate – rather, compactification of spheroid occurs.
In addition to assessment of changes in spheroid size or appearance upon treatment of cells with various compounds of interest (such as chemotherapeutic or targeted drugs, endocrine disruptors, etc.) or ionizing radiation, other studies can be carried out in spheroids. For instance, propidium iodide staining can be used to establish issues with cell plasma membrane integrity and the size of necrotic/late apoptotic cell population in a spheroid. In addition, spheroids can be fixed, immunostained, and mounted into microscopy chambers for high-resolution imaging. Furthermore, production of spheroids on a larger scale is possible using modified ULA surfaces with multiple cavities, thus providing sufficient amount of material for transcriptomic or proteomic studies.
Figure 2. Staining of Erbb2/HER2 in live MCF7 spheroids using the fluorescently labeled antibody. The spheroids were stained at 72 h post-seeding with 5 nM trastuzumab-Alexa647 and imaged after 1-h incubation and 3 washes with HBSS. BF indiates bright-field and HER2 indicates Alexa647 channel (Cytation 5 BioTek).