3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained methods
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The pellet culture method enabled us to modulate spheroid dimension by varying the number of cells in the starting unicellular suspension. In particular, for A549 cell line we obtained spheroids with a diameter of 800-900 µm starting from a cellular suspension of 200,000 cells. We also obtained compact cellular aggregates within 24 hours of the initial centrifugation using this method. However, the high number of vials centrifuge tubes needed to obtain sufficient spheroids (one spheroid/tube) to fill a 96- or 384-well plate (one spheroid/well) commonly used for high-throughput cytotoxicity screening tests made the method unmanageable. The RCCS method permitted us to obtain a higher number of large spheroids starting from a relatively small number of cells. With regard to A549 cell line, we seeded 40 × 10 6 cells in a single 50-ml vessel, obtaini...
Three-Dimensional Cell Culture Methods
A rotatory cell culture system (RCCS) (Synthecon Inc., Houston, TX, USA) was used. The rotator bases were placed inside a humidified 37 °C, 5% CO2 incubator and connected to power supplies set up externally to the incubator. All procedures were performed in sterile conditions under a laminar flow hood. Single cell suspensions of about 1 × 10 6 cells/ml of A549 were placed in the 50-ml rotating chamber at an initial speed of 12 rpm. As the majority of cells formed aggregates and these aggregates gradually enlarged, speed was increased over time to avoid aggregate sedimentation within the culture vessels which could hinder complete spheroid formation. The culture medium was changed every 4 days and tumor spheroids with an equivalent diameter ranging from about 500-1300 µm (depending on the cell line used) were obtained in around 15 day...
Hanging drop culture method
A549 cells were cultured in hanging drops using Perfecta3D™ Hanging Drop Plates (3DBiomatrix, Inc., Ann Arbor, MI, USA) or the GravityPLUS TM kit (InSphero AG, Schlieren, Switzerland), according to the manufacturers' instructions. Cells were seeded at various cell densities to verify the possibility of obtaining spheroids of at least 500-600 µm in diameter (2 × 10 3, 4 × 10 3, and 6 × 10 3 cells/well). In both assays, spheroidal colonies grew on the bottom of the wells after about 7 days' culture at 37 °C in atmosphere containing 5%CO 2.
Magnetic levitation method
The magnetic levitation method was used to obtain spheroids, as previously described by Haisler et al.. Briefly, cells were cultured in advance to confluence (at least 70-80%) in 2D and, on the day before the start of the experiment, they were incubated with an 8 µl/cm 2 magnetic nanoparticle assembly (Nano3D Biosciences Inc., Houston, Texas, USA) overnight to allow for cell attachment to the magnetic nanoparticles. The following day, the cells were detached and resuspended in medium in 24-well low-attachment culture plates (Corning Inc., Corning, NY,USA). In our study, different aliquots (1.5 × 10 3, 3 × 10 3, 6 × 10 3, 12 × 10 3 and 24 × 10 3 cells/well) of cells were seeded to define the optimal cell density needed to obtain spheroids with a diameter of about 500-600̴...
Microscopy and Image Analysis
For live imaging, spheroids of MRC-5 cell line were stained for DNA with Hoechst 33342 (Molecular Probes™). In particular, we exposed the spheroids to a solution of PBS 1x supplemented with Hoechst 33342 1 µg/ml. Viability of the A549 spheroids was measured with the LIVE/DEAD® Cell Viability Assay (Molecular Probes™). Briefly, samples were washed with warm phosphate-buffered saline (PBS), added to an ethidium-calcein mixture, and incubated for 30 min in a 37 °C incubator. After washing again with PBS, the spheroids were ready for the imaging procedure. All spheroids were mixed with low-melt agarose solution (Carl Roth GmbH) and the mixture was sucked into glass capillaries, with inner diameter of 1 mm. The agarose was allowed to gel at room temperature for five minutes before imaging.
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The pellet culture method enabled us to modulate spheroid dimension by varying the number of cells in the starting unicellular suspension. In particular, for A549 cell line we obtained spheroids with a diameter of 800-900 µm starting from a cellular suspension of 200,000 cells. We also obtained compact cellular aggregates within 24 hours of the initial centrifugation using this method. However, the high number of vials centrifuge tubes needed to obtain sufficient spheroids (one spheroid/tube) to fill a 96- or 384-well plate (one spheroid/well) commonly used for high-throughput cytotoxicity screening tests made the method unmanageable. The RCCS method permitted us to obtain a higher number of large spheroids starting from a relatively small number of cells. With regard to A549 cell line, we seeded 40 × 10 6 cells in a single 50-ml vessel, obtaining 200-250 spheroids ranging from 500-1100 µm in equivalent diameter after 15 days.
A rotatory cell culture system (RCCS) (Synthecon Inc., Houston, TX, USA) was used. The rotator bases were placed inside a humidified 37 °C, 5% CO2 incubator and connected to power supplies set up externally to the incubator. All procedures were performed in sterile conditions under a laminar flow hood. Single cell suspensions of about 1 × 10 6 cells/ml of A549 were placed in the 50-ml rotating chamber at an initial speed of 12 rpm. As the majority of cells formed aggregates and these aggregates gradually enlarged, speed was increased over time to avoid aggregate sedimentation within the culture vessels which could hinder complete spheroid formation. The culture medium was changed every 4 days and tumor spheroids with an equivalent diameter ranging from about 500-1300 µm (depending on the cell line used) were obtained in around 15 days. After the formation of the spheroids, the operator, working under the sterile laminar flow hood, transferred spheroids to 96-well low-attachment culture plates (Corning Inc., Corning, NY, USA) (one spheroid/well), each well previously filled with 100 µl of fresh culture medium.
A549 cells were cultured in hanging drops using Perfecta3D™ Hanging Drop Plates (3DBiomatrix, Inc., Ann Arbor, MI, USA) or the GravityPLUS TM kit (InSphero AG, Schlieren, Switzerland), according to the manufacturers' instructions. Cells were seeded at various cell densities to verify the possibility of obtaining spheroids of at least 500-600 µm in diameter (2 × 10 3, 4 × 10 3, and 6 × 10 3 cells/well). In both assays, spheroidal colonies grew on the bottom of the wells after about 7 days' culture at 37 °C in atmosphere containing 5%CO 2.
The magnetic levitation method was used to obtain spheroids, as previously described by Haisler et al.. Briefly, cells were cultured in advance to confluence (at least 70-80%) in 2D and, on the day before the start of the experiment, they were incubated with an 8 µl/cm 2 magnetic nanoparticle assembly (Nano3D Biosciences Inc., Houston, Texas, USA) overnight to allow for cell attachment to the magnetic nanoparticles. The following day, the cells were detached and resuspended in medium in 24-well low-attachment culture plates (Corning Inc., Corning, NY,USA). In our study, different aliquots (1.5 × 10 3, 3 × 10 3, 6 × 10 3, 12 × 10 3 and 24 × 10 3 cells/well) of cells were seeded to define the optimal cell density needed to obtain spheroids with a diameter of about 500-600 µm. A magnetic drive was then placed atop the well plate to levitate the cells to the air-liquid interface, where the cells aggregated and interacted to form large 3D structures. After 4-5 days' culture at 37 °C in an atmosphere containing 5% CO 2, the spheroids were f...
For live imaging, spheroids of MRC-5 cell line were stained for DNA with Hoechst 33342 (Molecular Probes™). In particular, we exposed the spheroids to a solution of PBS 1x supplemented with Hoechst 33342 1 µg/ml. Viability of the A549 spheroids was measured with the LIVE/DEAD® Cell Viability Assay (Molecular Probes™). Briefly, samples were washed with warm phosphate-buffered saline (PBS), added to an ethidium-calcein mixture, and incubated for 30 min in a 37 °C incubator. After washing again with PBS, the spheroids were ready for the imaging procedure. All spheroids were mixed with low-melt agarose solution (Carl Roth GmbH) and the mixture was sucked into glass capillaries, with inner diameter of 1 mm. The agarose was allowed to gel at room temperature for five minutes before imaging.
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