Reagent kit for investigating spheroid cell proliferation and/or viability
Catalog Number 3511-096-K
Current in vitro tumor models lack either a physiological context and/or reproducible format for evaluating tumor cells in vitro. At present, the most popular method for compound screening and pathway analysis involves culturing cancer cells on rigid, tissue culture treated plastic surfaces where the cells adhere non-specifically and proliferate as a monolayer, and as a result, these cells lose both morphology and gene expression profiles associated with tumors in vivo. Alternatively, single cell suspensions may be embedded in extracellular matrix (ECM) hydrogels to construct 3-D cultures; however, the resulting structures are dispersed throughout the gel and exhibit significant variability in morphology and size, limiting the establishment of physiological gradients and adversely affecting the reproducibility of each assay.
To address issues of reproducibility and to build more physiological tumor systems, well-established methods for multicellular spheroid formation were incorporated into 3-D culture models. Researchers have been using spheroid cultures for cancer research for over 40 years;1-3 however, there have been limitations regarding which cell lines could spontaneously form spheroids. For spontaneously spheroid assembly, it was shown that the cells produce an ECM that is deposited on the outer surface of the spheroid and that cell lines that could not spontaneously form compact spheroids were deficient or lacking in the formation of this ECM.4,5 It was later shown that the addition of ECM proteins to non-spheroid forming cells induced spontaneous spheroid formation, making the spheroid format compatible with most solid cancer cell models6. We have optimized this process, providing the necessary reagents to evaluate your cells using this method. Simply harvest cells, resuspend in spheroid formation ECM, and then culture in a 96 well spheroid formation plate. Spheroids generally form in 48 to 72 hours. Cell number and culture time determines spheroid size, and since each well produces one spheroid, researchers have complete control over spheroid dimensions with virtually no well to well variability.
For most tumor models, we recommend spheroids between 400 and 500 μm in diameter. This is sufficient to establish physiological gradients for nutrients, oxygen, pH, and catabolites due to limitations in diffusion through the multicellular layers. Another effect of these gradients is the establishment of heterogeneous cell populations with necrotic cells in the core, quiescent cells in the deeper layers, and proliferating cells on the spheroid surface; all of these factors reminiscent of an avascular tumor.7-10 Once formed, these multicellular tumor cell aggregates can be treated with pharmacological compounds to evaluate the effect on tumor spheroid growth; alternatively, specific genes or pathways may be manipulated to evaluate their effect on expansion of the in vitro tumor. This process can be monitored in real-time and label-free using image analysis software to measure spheroid area, and the kit is supplied with the colorimetric cell viability reagent MTT for quantitative end point analysis. The MTT tetrazolium ring is cleaved by mitochondrial dehydrogenases yielding purple formazan crystals which are subsequently solubilized for quantitation using Detergent Reagent.11
The 3-D Spheroid Colorimetric Proliferation/Viability Assay provides a useful tool for modeling tumor response in vitro. The kit uses 3-D Culture Qualified 96 Well Spheroid Formation Plate alongside a specialized Spheroid Formation ECM to drive aggregation and/or spheroid formation of cells. Upon completion of spheroid formation, the spheroid may be treated with pharmacological agents to evaluate tumor viability after drug treatment. Tumor spheroid expansion is visualized microscopically and can be quantitated through image analysis software for real-time and label free evaluation. At the conclusion of the assay, cell viability may be assessed by absorbance using MTT. The 3D Spheroid Colorimetric Proliferation/Viability Assay offers an in vitro, standardized, three dimensional, high-content format for inducing multicellular tumor spheroid (MCTS) formation and quantitating cell viability within the spheroids in response to pharmacological treatment.
Figure 1.Steps comprising the 3-D spheroid colorimetric proliferation/viability assay.
For Research Use Only. Not for use in diagnostic procedures.
Perform these procedures in a biological hood using aseptic technique to prevent contamination.
A. Cell Harvesting
Culture cells per manufacturer’s recommendation. The following procedure is suggested and may need to be optimized to suit the cell type(s) being studied.
B. 3-D Spheroid Colorimetric Proliferation/Viability Assay
Figure 2.Process for analysis of spheroid expansion. A) Capture image and convert to 8 bit; B) Set threshold to capture the total structure; C) Select structure to calculate total area.
Figure 3.Spheroid growth of MDA-MB-231 breast cancer cells. Cells were seeded at 3,000 cells/well in the presence of spheroid formation ECM and incubated for 72 hours at 37 °C, 5% CO2 to induce spheroid formation. At that time, 50 µL of complete medium was added to each well, and spheroids were incubated at 37 °C, 5% CO2. Spheroids were photographed every 24 hours and images were analyzed using ImgaJ software.
First the areas were calculated for each condition, and these values were used to generate average and standard deviations:
Next the area for each spheroid before treatment was subtracted to determine spheroid expansion.
Finally, the resulting areas were divide by the area obtained for the no inhibitor control, providing the proliferation index as a percentage of control:
First, the absorbance was measured at 570 nm for each well, and the average and standard deviations were calculated for each sample using Excel:
Next, the background was subtracted from each sample, and the data was plotted with a trendline to evaluate the relationship between cell seeding density and absorbance (570 nm):
First, the absorbance at 570 nm was measured for each well, and the average and standard deviations were calculated for each sample using Excel:
Next, the background was subtracted from each sample:
Finally, the background-corrected absorbance was divided by the value obtained for the no inhibitor control, providing the viability index as a percentage of control:
Using the linear portion of the inhibition curve, the IC50 may be estimated:
IC50 ~ 15 µM for MDA-MB-231 Spheroids
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