Assays and Tools to Monitor Biology in 3D Cell Cultures
Researchers are turning to 3D culture methods such as spheroids and organoids to create model systems for studying cellular biology. 3D cell culture mimics the tissue or tumor environment more closely than a 2D environment by facilitating more natural interactions with a matrix and even allowing cells to secrete their own matrix. 3D cell culture also offers more natural cell-to-cell contacts and produces a gradient of access to O2 and nutrients.
Often, 3D cell culture can reveal dramatic differences in cellular responses compared to monolayer culture. Monitoring the biology of cells in 3D culture can be approached by looking at changes in cell health, metabolism and expression. Ultimately, these phenotypic changes are related back to the genotype of the cell or the alterations in the genotype that led to a disease state. We offer assays and tools to address each step in your workflow, from cell health and metabolic changes to gene expression and analysis.
CellTiter-Glo® 3D Cell Viability Assay
Monitor cell viability through measurement of ATP with a reagent specifically designed for 3D cultures. Utilizes the same ATP measurement luciferase chemistry of the classic CellTiter-Glo® Assay with greater lytic power, which allows deeper penetration yielding greater ATP release for quantitation. Co-measure dead cells with same-well multiplexing with CellTox™ Green Cytotoxicity assay or media sampling with the LDH-Glo™ Cytotoxicity Assay.
Lytic Capacity of CellTiter-Glo® 3D Compared to ATPlite 1Step Reagent
Drug Screening Using Single Organoids
Organoids provide an essential tool for more accurately predicting in vivo responses from drug screening campaigns as they are more representative of the cellular heterogeneity within human tissue. Despite the pressing need for such models in drug screening, adoption of organoid in vitro assays for HTS is still limited due to workflow challenges, including lack of reproducibility and scalability. Highlighted here, Cell Microsystems and Promega provide an automated solution for reproducibly generating hundreds of organoids ready for use in plate-based assays that have been optimized and validated for 3D cell culture models.
Viability of unsorted compared to sorted organoids.
Energy metabolism is critical for cellular health and function and metabolites are linked to cellular energy, creation of cellular building blocks and signaling pathways. We offer several assays to measure metabolic activity with optimized protocols for 3D cell culture applications, including lactate, glutamine, oxidative stress and dinucleotide detection assays. Common sample preparation allows co-measurement with Glucose-Glo™, Lactate-Glo™, Glutamate-Glo™ and Glutamine/Glutamate-Glo™ Assays from the same sample.
Sensitive bioluminescent measure of glucose. Requires only 2–5μl of conditioned cell culture media per time point allowing multiple samples per well for time course studies. Tested on microspheroid culture conditioned media.
Insulin-Mediated Inhibition of Gluconeogenesis of iCell® Hepatocyte Spheroids
Non-Lytic Assay Option
Monitor glutamate changes in 3D cultures using the non-lytic, sensitive bioluminescent assay option. Assay requires on 2–5μl of culture media per time point allowing multiple samples from the same well. Cells may be used for other cell-based assays or nucleic acid isolation.
Non-Lytic Assay Option
The Glycerol-Glo™ Assay is a bioluminescent assay for rapid and sensitive measurement of glycerol in a variety of biological samples including cells cultured in 3D. Glycerol is often measured as the product of lipolysis, where it is released from triglycerides. Any processes that result in changes in glycerol concentration, both extracellular and intracellular, can be studied with the Glycerol-Glo™ Assay.
Cells grown in 3D culture can have differences in gene expression when compared to monolayer cultures. The changes in cell-to-cell contacts and cell-to-matrix contacts will be very different and the gradient of oxygen and nutrients within the culture will also influence gene expression. To analyze differences in gene expression, RNA must be isolated and quantified. Then, specific genes are analyzed by RT-qPCR or cellular changes are monitored through techniques like RNA-seq.
ReliaPrep™ RNA Miniprep Systems
Isolate application-ready total RNA or miRNA from 3D cultures to monitor changes in expression. The ReliaPrep™ RNA Miniprep Systems synergize with non-lytic cellular assays like the RealTime-Glo™ MT Cell Viability Assay.
Extraction of RNA from HEK293 Microtissue Spheroids
Understanding genotype-phenotype or phenotype-genotype differences is important, especially when studying primary cells or tumors. You may analyze a genotype and use 3D cell culture to understand the phenotype or you may choose to study the phenotype and then look at the genotype to understand why these changes occur. For genomic DNA analysis, you will need to extract and quantify the DNA. Then you will look at specific genes through qPCR or choose to examine the genome with NGS. If you are working with primary cells, you must be able to differentiate which sample came from which donor—STR profiles are often chosen for that task. The STR profile is also important for confirming that the cells you are working with are truly the cells you desire.
Maxwell® RSC Cultured Cells DNA Kit
Isolate application-ready genomic DNA from 3D cultures for genomic analyses with a simple, walk-away automated protocol using a Maxwell® RSC Instrument. Purified DNA is ready for analysis in about 45 minutes. Isolated gDNA can be used for qPCR-based analyses for gene copy number and biomarkers, STR-based analysis for sample tracking or verification, and sequencing-based analyses.