Figure 1. Detection of viable cells in Cell-Mate3D matrix using the LIVE/DEAD viability assay. AU565 breast cancer cells growing in Cell-Mate3D matrix were stained using the Invitrogen LIVE/DEAD Viability/Cytotoxicity Kit and counterstained using Invitrogen NucBlue Live ReadyProbes Reagent. (A) In the untreated sample, live cells fluoresce green and nuclei fluoresce blue; very few nonviable (red-fluorescent) cells are detected. (B) After treatment with 0.5% Triton X-100 detergent, the sample contains primarily nonviable (red-fluorescent) cells; the purple color of the cells in the image represent the overlap of the red- and blue-fluorescent stains. Samples were imaged using an inverted confocal microscope at 20x magnification.
Fluorescent Reagents for Staining 3D Cell Cultures Created With Cell-Mate3D Matrix
Cancer biology in the third dimension
Unlike their 2D tissue culture counterparts, tumors and their associated microenvironments contain a highly complex and dynamic set of interactions between different cell types , multiple chemical gradients , and a variety of extracellular matrix components [3,4]. In addition to the biochemical reactions that take place, a tumor’s physical characteristics, including the rigidity or stiffness of the extracellular matrix, can regulate its physiology [5–7]. The next discoveries in cancer biology and treatment will require tools that help to create more relevant and predictive models as well as methods to effectively analyze them.
The combination of 3D cell culture techniques with methods for analyzing cell health promises to deliver new insights into cancer mechanisms and new strategies for interrupting or subverting critical pathways. Here we describe the application of fluorescence-based assays and protocols to a complex 3D environment, often with only minor adjustments to exposure time or reagent concentration. Fluorescent reagents from Thermo Fisher Scientific can be used to assess cell function—including viability, proliferation, and apoptosis—in response to various 3D culture conditions and treatments.
Create 3D cultures with Cell-Mate3D matrix
Cell-Mate3D™ matrix from BRTI Life Sciences is a tissue-like matrix that offers researchers a biologically relevant and chemically defined microenvironment for in vitro and in vivo biomedical research. The Cell-Mate3D matrix is composed of two naturally occurring biopolymers: chitosan, a positively charged polysaccharide derived from chitin, found in the exoskeleton of crustaceans; and hyaluronic acid, a linear polysaccharide found in the extracellular matrix of connective, epithelial, and neural tissues. Hyaluronic acid is typically involved in cell proliferation, migration, embryonic development, and wound healing.
When Cell-Mate3D Dry Blend is combined with cells resuspended in Cell-Mate3D Hydration Fluid, the matrix forms instantly through electrostatic interactions between carboxyl groups on the hyaluronic acid and the amine groups on the chitosan. The resulting poly-electrolytic complex is a fibrous matrix that exhibits tissue-like stiffness, emulating the natural cell environment. The Cell-Mate3D matrix enables modeling of cell migration and proliferation; laboratory techniques such as cell staining and imaging, flow cytometry, and scanning electron microscopy can be used with this technology .
Determine cell viability in 3D cultures
The Invitrogen LIVE/DEAD Viability/Cytotoxicity Kit provides a well-established fluorescence assay for determining viability in a wide range of animal cells. The LIVE/DEAD viability assay comprises two fluorescent probes—calcein AM, a cell-permeant esterase substrate, and ethidium homodimer-1, a cell-impermeant high-affinity nucleic acid stain—that are used simultaneously to stain cells. We used the LIVE/DEAD viability assay with 3D cell cultures that were created by embedding AU565 breast cancer cells into Cell-Mate3D matrix. After the matrix with embedded cells was incubated for 4 days, one sample was left untreated and an equivalent sample was treated with 0.5% Triton X-100 detergent, which disrupts cell membranes and kills cells. Both samples were then assayed using the LIVE/DEAD Viability/Cytotoxicity Kit and the supplied protocol, except that the incubation time was reduced from 30 to 20 minutes to optimize staining levels in this system. After counterstaining with a blue-fluorescent nucleic acid dye, the samples were imaged on an inverted confocal microscope (Figure 1). The untreated sample shows live cells labeled with green fluorescence, whereas the treated sample shows dead cells labeled with red fluorescence.
Detect cell proliferation in 3D cultures
In addition to its essential role in development, cell proliferation is an important marker of cancer cells and can serve as a target of anti-cancer therapies. The Invitrogen Click-iT EdU Imaging Kits provide a simple, efficient proliferation assay that detects DNA synthesis using fluorescence microscopy. In the Click-iT EdU assay, a modified thymidine analog (5-ethynyl-2´-deoxyuridine, or EdU) is introduced into cells, incorporated into newly synthesized DNA, and then labeled with a brightly fluorescent Invitrogen Alexa Fluor dye in a fast, highly specific click reaction. We used the Click-iT EdU Alexa Fluor 488 Imaging Kit (with no changes to the supplied protocol) with 3D cell cultures that were created by embedding HeLa cells into Cell-Mate3D matrix, and found that proliferating cells (which fluoresce green) were easily visualized in 4-day-old cultures (Figure 2).
Figure 2. Detection of proliferating cells in the Cell-Mate3D matrix using Click-iT EdU staining. HeLa cells were embedded in the Cell-Mate3D matrix and cultured for 4 days. Proliferating cells were detected using the Invitrogen Click-iT EdU Alexa Fluor 488 Imaging Kit (green). Cells were counterstained with the Invitrogen NucBlue Fixed Cell ReadyProbes Reagent (blue), and imaged using an inverted confocal microscope at 60x magnification.
Detect apoptosis in 3D cultures
The Invitrogen CellEvent Caspase-3/7 Green Detection Reagent enables quick and reliable quantitation of apoptotic cells in culture. This nonfluorescent substrate is composed of the 4–amino acid peptide DEVD, which contains the caspase-3/7 recognition site, conjugated to a nucleic acid–binding dye. In the presence of activated caspase-3 or -7, the substrate is cleaved, freeing the dye to bind to DNA and producing a bright green-fluorescent signal indicative of apoptosis. To test this reagent with 3D cell cultures, we embedded AU565 breast cancer cells in CellMate-3D matrix and cultured the cells for 12 days. During this time, one sample was left untreated and an equivalent sample was treated with 100 μM paclitaxel (generic name of Taxol pharmaceutical) to induce apoptosis. Samples were then stained with 15 μM CellEvent Caspase-3/7 reagent (3 times the recommended concentration). Green-fluorescent apoptotic cells were clearly seen in the paclitaxel-treated sample by confocal microscopy (Figure 3).
Figure 3. Detection of apoptotic cells in the Cell-Mate3D matrix using CellEvent Caspase-3/7 Green reagent. AU565 breast cancer cells were embedded in the Cell-Mate3D matrix and cultured for 12 days. During this time, cells were (A) left untreated or (B) treated with 100 μM paclitaxel to induce apoptosis. Cells were then incubated with the Invitrogen CellEvent Caspase-3/7 Green Detection Reagent (15 μM) for 30 min to label apoptotic cells with green fluorescence, counterstained with the Invitrogen NucBlue Live ReadyProbes Reagent (blue), and imaged using an inverted confocal microscope at 20x magnification.
Apply fluorescence protocols to your 3D experiments
Although some optimization was required when applying fluorescent probes to 3D cell cultures growing in Cell-Mate3D matrix, we found that generally the reagents were able to penetrate the complex environment and accurately report cell health parameters and enzyme activity. Analysis of cell function within 3D cell cultures will greatly benefit from the plethora of fluorescent cell function probes available.
Acknowledgments: This article was contributed by Beth Lindborg, Caitlin Johnson, Yi Wen Chai, Eu Han Lee, and Scott Brush, BRTI Life Sciences, Two Harbors, Minnesota (brtilifesciences.com). Images were acquired at the University of Minnesota-University Imaging Center (UMN-UIC) facility; we greatly appreciate UMN-UIC member Grant Barthel’s assistance with confocal microscopy.
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