Production and Multi-Parameter Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM) of Multicellular Spheroids
August 9th, 2024
Here, we describe different multicellular spheroid formation methods to perform follow-up multi-parameter live cell microscopy. Using fluorescence lifetime imaging microscopy (FLIM), cellular autofluorescence, staining dyes, and nanoparticles, the approach for analysis of cell metabolism, hypoxia, and cell death in live three-dimensional (3D) cancer and stem cell-derived spheroids is demonstrated.
In this work, we present and compare spheroid formation methods that can be used for analysis of cell metabolism and oxygen distribution using live cell microscopy. In recent years, fluorescence lifetime imaging microscopy has been extensively used to investigate metabolic biomarkers like NADPH and FAD in live cells, and several fluorescent nanoparticles have been developed to multiplex such measurements with imaging of cell and tissue oxygenation. Multicellular spheroids, organoids, and organ-on-a-chip can replicate a complex, in-vivo-like microenvironment, minimizing the need in animal research.
For spheroid production, we show different formation methods, can span from low to high throughput. They also highlight their optical accessibility, compatibility with fluorescence lifetime imaging microscope, and the possibility of including extracellular matrix components. So, even though 3D in vitro models provide better context in comparison to 2D cultures, their high variability, low reproducibility, and incomplete experimental reporting remain a problem.
Parameters such as the spheroid size, the nutrient composition, the extracellular viscosity, and even spheroid formation methods can all lead to increased cellular heterogeneity. With this protocol, we aim to harmonize and standardize the spheroid production methods, highlighting key aspects important for life-continuous and multiparametric analysis of spheroids using FLIM microscopy.
Overview
This article presents various methods for forming multicellular spheroids to analyze cell metabolism and oxygen distribution using live cell microscopy. The study highlights the use of fluorescence lifetime imaging microscopy (FLIM) to investigate metabolic biomarkers in live cells.
Key Study Components
Area of Science
- Neuroscience
- Cell Biology
- Live Cell Imaging
Background
- Multicellular spheroids and organoids replicate in-vivo-like microenvironments.
- Fluorescence lifetime imaging microscopy is used to study metabolic biomarkers.
- High variability and low reproducibility are challenges in 3D in vitro models.
- Different spheroid formation methods can be employed for various applications.
Purpose of Study
- To compare spheroid formation methods for live cell microscopy.
- To analyze cell metabolism, hypoxia, and cell death in 3D models.
- To improve the understanding of oxygen distribution in multicellular systems.
Methods Used
- Fluorescence lifetime imaging microscopy (FLIM)
- Cellular autofluorescence analysis
- Use of staining dyes and fluorescent nanoparticles
- Different spheroid formation techniques
Main Results
- Demonstrated methods for analyzing cell metabolism in 3D spheroids.
- Showed compatibility of spheroids with FLIM for metabolic studies.
- Highlighted the importance of extracellular matrix components in spheroid formation.
- Addressed challenges in reproducibility and variability of 3D cultures.
Conclusions
- Different spheroid formation methods can enhance live cell analysis.
- FLIM provides valuable insights into metabolic processes in 3D models.
- Continued development of these methods can reduce reliance on animal models.
Frequently Asked Questions
Chapters in this video
0:00
Introduction
1:51
High‐Throughput Generation of Multicellular Spheroids from Human Cancer Cell Line for Live 3D Imaging
4:15
Assessing the Impact of Spheroid Formation Methods in 3D Cell Culture Systems Using Live Cell Fluorescence Lifetime Imaging Microscopy (FLIM)
