Rapid Clearing for High Resolution 3D Imaging of Ex Vivo Pancreatic Cancer Spheroids

Abstract

The currently accepted imaging methods have been a central hurdle to imaging the finer details of tumor behavior in three-dimensional (3D) ex vivo multicellular culture models. In our search for an improved way of imaging tumor behavior in its physiological-like niche, we developed a simple, efficient, and straightforward procedure using standard reagents and imaging equipment that significantly enhanced 3D imaging up to a ~200-micron depth. We tested its efficacy on pancreatic spheroids, prototypes of high-density tissues that are difficult to image. We found we could both save time with this method and extract information about pancreatic tumor spheroids that previously was difficult to obtain. We were able to discern clear differences in the organization of pancreatic tumor spheroids generated from different origins, suggesting cell-specific, inherent, bottom-up organization with a correlation to the level of malignancy. We also examined the dynamic changes in the spheroids at predetermined time points, providing important information related to tissue morphogenesis and its metabolic state. Lastly, this process enabled us to assess a drug vehicle’s potential to penetrate dense tumor tissue by improving our view of the inert particles’ diffusion in the 3D spheroid. This clearing method, a simple procedure, can open the door to more accurate imaging and reveal more about cancer behavior.

Electrostatic Assembly of Multiarm PEG-Based Hydrogels as Extracellular Matrix Mimics: Cell Response in the Presence and Absence of RGD Cell Adhesive Ligands

“…With the 98% RGD polymer, spheroids of size 200 μm or more exhibited in vivo-like necrotic cores; , therefore, the rest of studies we focused on using the unmodified polymers and polymers with 98% RGD conjugation will henceforth be now referred to as RGD-hydrogel.…”

Gene Electrotransfer Efficiency in 2D and 3D Cancer Cell Models Using Different Electroporation Protocols: A Comparative Study

“…Indeed, our results assessed that the application of electric field (ECT, GET and HV-LV protocols) affected the growth of spheroids ( Figure 7 ) and contribute to the detachment of cells on spheroids’ surface (images Figure 8 ). The method of clearing optimized on spheroids [ 41 , 42 , 43 ] and on 3D tissues in a more general way [ 44 ] allowed us to confirm that all cells constituting the spheroids underwent electropermeabilization, regardless of the protocols applied. The same tissue-clearing procedure was used in our experiments to study DNA transfection in deeper layers.…”

Analyzing force measurements of multi-cellular clusters comprising indeterminate geometries

“…Spheroids were prepared as detailed in our previous work (Steinberg et al 2020 ). Master 3D Petri dish 35-well arrays (Microtissues Inc., Providence, RI, USA) were used to create micro-wells made of 2% UltraPureTM agarose hydrogel (Invitrogen, Carlsbad, CA, USA).…”