Abstract
Multicellular spheroids have served as a promising preclinical model for drug efficacy testing and disease modeling. Many microfluidic technologies, including those based on water–oil–water double emulsions, have been introduced for the production of spheroids. However, sustained culture and the in situ characterization of the generated spheroids are currently unavailable for the double emulsion-based spheroid model. This study presents a streamlined workflow, termed the double emulsion-pretreated microwell culture (DEPMiC), incorporating the features of (1) effective initiation of uniform-sized multicellular spheroids by the pretreatment of double emulsions produced by microfluidics without the requirement of biomaterial scaffolds; (2) sustained maintenance and culture of the produced spheroids with facile removal of the oil confinement; and (3) in situ characterization of individual spheroids localized in microwells by a built-in analytical station. Characterized by microscopic observations and Raman spectroscopy, the DEPMiC cultivated spheroids accumulated elevated lipid ordering on the apical membrane, similar to that observed in their Matrigel counterparts. Made possible by the proposed technological advancement, this study subsequently examined the drug responses of these in vitro-generated multicellular spheroids. The developed DEPMiC platform is expected to generate health benefits in personalized cancer treatment by offering a pre-animal tool to dissect heterogeneity from individual tumor spheroids.
Highlights
The success rates of cancer clinical trials remain relatively low despite substantial progress made in the past decades[1]
Microfluidics-generated double emulsions (DEs) have been employed as a pretreatment to encourage spheroid initiation, whereas microwell culture made the localization of spheroids possible for subsequent in situ analysis
Calu-3 cells cultivated by the double emulsion-pretreated microwell culture (DEPMiC) method exhibited cyst structures after 1 day of pretreatment in DEs and the circular morphology resembled the phenomena observed in their Matrigel counterparts
Summary
The success rates of cancer clinical trials remain relatively low despite substantial progress made in the past decades[1]. Cancer is notoriously complex and is characterized by profound heterogeneity between patients and even between malignant cells within a single tumor[3,4]. In response to this complexity, most efforts have focused on cancer genomics and analyzing. Efforts to overcome these obstacles have led to the development of in vivo-mimicking three-dimensional. Mounting evidence has demonstrated that ex vivo cultivated tumor spheroids behave very differently from monolayer cultured cells and reflect many features of solid tumors[8,9,10]. The 3D construct of spheroids resembles solid tumors by the presence of a central necrotic and hypoxic core, which plays an important role in initiating the signaling pathway and transcriptional alterations to promote cancer cell survival, as well as the generation of cancer stem cells, two factors that are considered to be closely associated with drug resistance and tumor relapse[12]
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