Bioluminescence Imaging of Spheroids for High‐throughput Longitudinal Studies on 3D Cell Culture Models

Abstract

Bioluminescent (BL) cell-based assays based on two-dimensional (2D) monolayer cell cultures represent well-established bioanalytical tools for preclinical screening of drugs. However, cells in 2D cultures do not often reflect the morphology and functionality of living organisms, thus limiting the predictive value of 2D cell-based assays. Conversely, 3D cell models have the capability to generate the extracellular matrix and restore cell-to-cell communications; thus, they are the most suitable model to mimic in vivo physiology. In this work, we developed a nondestructive real-time BL imaging assay of spheroids for longitudinal studies on 3D cell models. A high-throughput BL 3D cell-based assay in micropatterned 96-well plate format is reported. The assay performance was assessed using the transcriptional regulation of nuclear factor K beta response element in human embryonic kidney (HEK293) cells. We compared concentration-response curves for tumor necrosis factor-α with those obtained using conventional 2D cell cultures. One of the main advantages of this approach is the nonlysing nature of the assay, which allows for repetitive measurements on the same sample. The assay can be implemented in any laboratory equipped with basic cell culture facilities and paves the way to the development of new 3D bioluminescent cell-based assays.

Bioluminescence‐based cytotoxicity assay for simultaneous evaluation of cell viability and membrane damage in human hepatoma HepG2 cells

“…We have recently demonstrated that the decrease of luminescence intensity of beetle luciferase expressed under the control of a constitutive promoter shows good agreement with cell viability determined by the neutral red uptake assay . Furthermore, it has been reported that beetle luciferases, including firefly luciferase from Photinus pyralis and click beetle luciferase from Pyrearinus termitilluminans (Emerald luciferase, ELuc), have been utilized for continuous long‐term cytotoxicity monitoring in three‐dimensional spheroids by repetitive non‐destructive bioluminescence measurements, by utilizing the high stability and permeability properties of their substrate, d ‐luciferin . On the other hand, endoplasmic reticulum (ER)‐targeted secretion‐type copepod luciferase from Gaussia princeps , in which the ER retention signal (Lys‐Asp‐Glu‐Leu; KDEL) is fused in‐frame to the extreme C‐terminus of GLuc (hereinafter referred to as GLuc‐KDEL), has been used for high‐sensitivity cytotoxicity monitoring by measuring the extracellular release of GLuc‐KDEL by damaged cells …”

“…[13] Furthermore, it has been reported that beetle luciferases, including firefly luciferase from Photinus pyralis and click beetle luciferase from Pyrearinus termitilluminans (Emerald luciferase, ELuc), [14,15] have been utilized for continuous long-term cytotoxicity monitoring in three-dimensional spheroids by repetitive non-destructive bioluminescence measurements, by utilizing the high stability and permeability properties of their substrate, D-luciferin. [16,17] On the other hand, endoplasmic reticulum (ER)-targeted secretion-type copepod luciferase from Gaussia princeps, [18] in which the ER retention signal (Lys-Asp-Glu-Leu; KDEL) is fused in-frame to the extreme C-terminus of GLuc (hereinafter referred to as GLuc-KDEL), has been used for high-sensitivity cytotoxicity monitoring by measuring the extracellular release of GLuc-KDEL by damaged cells. [19] Extracellular release of high mobility group box 1 (HMGB1), a representative of damage-associated molecular pattern (DAMP) molecules, from damaged cells is considered the key molecule in the evaluation of cytotoxicity potential of toxicant.…”

A Genetically Encoded Bioluminescence Intracellular Nanosensor for Androgen Receptor Activation Monitoring in 3D Cell Models

“…We first characterized NanoBiT re-assembly in HEK293 grown as a monolayer and in HEK293 spheroids with an average diameter of 130 ± 20 µm. This size was selected because it has been shown to be suitable for maintaining an adequate cell viability with a negligible necrotic core, of less than 2% of cells [23][24][25]. Since all BL systems require molecular oxygen, the availability of oxygen is a crucial factor in BL measurements.…”

Lab-on-Chip Platform for Culturing and Dynamic Evaluation of Cells Development

“…As the cells in their natural environment are constantly subjected to dynamic, various influences, this model do not imitate in vivo conditions accurately. According to other papers [4,5], cultures established in Petri dishes may be characterized by notable degradation of cells, changes in their metabolism, or gene expression.…”