Cell Biology

Three-dimensional (3D) cell culture models are widely used in tumor studies to more accurately reflect cell-cell interactions and tumor growth conditions in vivo. 3D anchorage-independent spheroids derived by culturing cells in ultra-low attachment (ULA) conditions is particularly relevant to ovarian cancer, as such cell clusters are often observed in malignant ascites of late-stage ovarian cancer patients. We and others have found that cells derived from anchorage-independent spheroids vary widely in gene expression profiles, proliferative state, and metabolism compared to cells maintained under attached culture conditions. This includes changes in mitochondrial function, which is most commonly assessed in cultured live cells by measuring oxygen consumption in extracellular flux assays. To measure mitochondrial function in anchorage-independent multicellular aggregates, we have adapted the Agilent Seahorse extracellular flux assay to optimize measurements of oxygen consumption and extracellular acidification of ovarian cancer cell spheroids generated by culture in ULA plates. This protocol includes: (i) Methods for culturing tumor cells as uniform anchorage-independent spheroids; (ii) Optimization for the transfer of spheroids to the Agilent Seahorse cell culture plates; (iii) Adaptations of the mitochondrial and glycolysis stress tests for spheroid extracellular flux analysis; and (iv) Suggestions for optimization of cell numbers, spheroid size, and normalization of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) values. Using this method, we have found that ovarian cancer cells cultured as anchorage-independent spheroids display altered mitochondrial function compared to monolayer cultures attached to plastic dishes. This method allows for the assessment of mitochondrial function in a more relevant patho/physiological culture condition and can be adapted to evaluate mitochondrial function of various cell types that are able to aggregate into multicellular clusters in anchorage-independence.

Graphic abstract:



Workflow of the Extracellular Flux Assay to Measure Respiration of Anchorage-independent Tumor Cell Spheroids.

The high attrition rate in drug development processes calls for additional human-based model systems. However, in the context of brain disorders, sampling live neuronal cells for compound testing is not applicable. The use of human induced pluripotent stem cells (iPSCs) has revolutionized the field of neuronal disease modeling and drug discovery. Thanks to the development of iPSC-based neuronal differentiation protocols, including tridimensional cerebral organoids, it is now possible to molecularly dissect human neuronal development and human brain disease pathogenesis in a dish. These approaches may allow dissecting patient-specific treatment efficacy in a disease-relevant cellular context. For drug discovery approaches, however, a highly reproducible and cost-effective cell model is desirable. Here, we describe a step-by-step process for generating robust and expandable neural progenitor cells (NPCs) from human iPSCs. NPCs generated with this protocol are homogeneous and highly proliferative. These features make NPCs suitable for the development of high-throughput compound screenings for drug discovery. Human iPSC-derived NPCs show a metabolism dependent on mitochondrial activity and can therefore be used also to investigate neurological disorders in which mitochondrial function is affected. The protocol covers all steps necessary for the preparation, culture, and characterization of human iPSC-derived NPCs.

Graphic abstract:

Schematic of the protocol for the generation of human iPSC-derived NPCs

Mitochondrial dysfunction is a principal feature of acute pancreatitis (AP) although the underlying mechanisms are still unclear. AP precipitants induce Ca²⁺-dependent formation of the mitochondrial permeability transition pore (MPTP) in pancreatic acinar cells (PACs), leading to ATP depletion and necrosis. Evaluations of mitochondrial bioenergetics have mainly been performed in isolated PACs using confocal microscopy, with assessment of mitochondrial membrane potential, NADH/FAD⁺ and ATP levels, coupled with patch-clamp electrophysiology. These studies are technically demanding and time-consuming. Application of Seahorse flux analysis now allows detailed investigations of bioenergetics changes to be performed in cell populations using a multi-well plate-reader format; rates of oxygen consumption (OCR) and extracellular acidification (ECAR) provide important information about cellular respiration and glycolysis, respectively. Parameters such as maximal respiration, ATP-linked capacity and proton leak can be derived from application of a respiratory function “stress” test that involves pharmacological manipulation of the electron transport chain. The use of Seahorse Flux analysis therefore provides a quick, and convenient means to measure detailed cellular bioenergetics and allows results to be coupled with other plate-reader based assays, providing a fuller understanding of the pathophysiological consequences of mitochondrial bioenergetics alterations.

Many Candida species, such as the opportunistic human pathogen Candida albicans, are Crabtree-Negative yeasts and are therefore highly dependent on the energy generated through oxidative phosphorylation. Respiration control is linked to a range of aspects of C. albicans cell physiology that appear to be important for virulence, most notably its ability to switch from yeast to hyphal forms and the maintenance of the cell wall. The following protocol allows for the measurement and characterization of respiration in C. albicans using high resolution respirometry. We outline how addition of respiration inhibitors can be used to assay the “mode” of respiration, mitochondrial health and the level of electron transport that is coupled to ATP synthase activity in living cell cultures. These data provide useful insight into the effects of external factors, such as exposure to anti-fungal compounds, or internal changes such as genetic alterations on respiratory performance.